Cyanine dyes

ABSTRACT

The invention provides a novel class of cyanine dyes that are functionalized with a linker moiety that facilitates their conjugation to other species. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS Claim of Priority

This application is a continuation of U.S. patent application Ser. No.12/652,625, filed Jan. 5, 2010, now U.S. Pat. No. 8,436,153, which is acontinuation of U.S. patent application Ser. No. 11/051,666 filed Feb.4, 2005, now U.S. Pat. No. 7,705,150, and claims priority under 35U.S.C. §119(e) to U.S. provisional application No. 60/542,137, filedFeb. 4, 2004, the specifications of which are incorporated herein byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates generally to the synthesis of fluorescentcompounds that are analogues of cyanine dyes. The compounds of theinvention are fluorophores that are derivatized to allow their facileattachment to a carrier molecule.

2. Background

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, saccharides, pharmaceuticals, metabolites,microorganisms and other materials of diagnostic value. Examples of suchmaterials include narcotics and poisons, drugs administered fortherapeutic purposes, hormones, pathogenic microorganisms and viruses,peptides, e.g., antibodies and enzymes, and nucleic acids, particularlythose implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity, which characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which is attached toone or more of the interacting materials. The specific labeling methodchosen often dictates the usefulness and versatility of a particularsystem for detecting an analyte of interest. Preferred labels areinexpensive, safe, and capable of being attached efficiently to a widevariety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations. However, suchlabels are expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance, and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest because of the large number of such labels that areknown in the art. Moreover, as discussed below, the literature isreplete with syntheses of fluorescent labels that are derivatized toallow their attachment to other molecules, and many such fluorescentlabels are commercially available.

In addition to being directly detected, many fluorescent labels operateto quench the fluorescence of an adjacent second fluorescent label.Because of its dependence on the distance and the magnitude of theinteraction between the quencher and the fluorophore, the quenching of afluorescent species provides a sensitive probe of molecular conformationand binding, as well as other interactions. An excellent example of theuse of fluorescent reporter quencher pairs is found in the detection andanalysis of nucleic acids.

Fluorescent nucleic acid probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. As information from the Human Genome Project accumulates, thelevel of genetic interrogation mediated by fluorescent probes willexpand enormously. One particularly useful class of fluorescent probesincludes self-quenching probes, also known as fluorescence energytransfer probes, or FET probes. The design of different probes usingthis motif may vary in detail. In an exemplary FET probe, both afluorophore and a quencher are tethered to a nucleic acid. The probe isconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing alternativemethods.

Probes containing a fluorophore-quencher pair have been developed fornucleic acid hybridization assays where the probe forms a hairpinstructure, i.e., where the probe hybridizes to itself to form a loopsuch that the quencher molecule is brought into proximity with thereporter molecule in the absence of a complementary nucleic acidsequence to prevent the formation of the hairpin structure (see, forexample, WO 90/03446; European Patent Application No. 0 601 889 A2).When a complementary target sequence is present, hybridization of theprobe to the complementary target sequence disrupts the hairpinstructure and causes the probe to adopt a conformation where thequencher molecule is no longer close enough to the reporter molecule toquench the reporter molecule. As a result, the probes provide anincreased fluorescence signal when hybridized to a target sequencecompared to when they are unhybridized

Assays have also been developed for detecting a selected nucleic acidsequence and for identifying the presence of a hairpin structure usingtwo separate probes, one containing a reporter molecule and the other aquencher molecule (see, Meringue, et al., Nucleic Acids Research, 22:920-928 (1994)). In these assays, the fluorescence signal of thereporter molecule decreases when hybridized to the target sequence dueto the quencher molecule being brought into proximity with the reportermolecule.

One particularly important application for probes that include areporter-quencher molecule pair is in nucleic acid amplificationreactions, such as polymerase chain reactions (PCR), to detect thepresence and amplification of a target nucleic acid sequence. Ingeneral, nucleic acid amplification techniques have opened broad newapproaches to genetic testing and DNA analysis (see, for example,Arnheim et al. Ann. Rev. Biochem., 61: 131-156 (1992)). PCR, inparticular, has become a research tool of major importance withapplications in, for example, cloning, analysis of genetic expression,DNA sequencing, genetic mapping and drug discovery (see, Arnheim et al.,supra; Gilliland et al., Proc. Natl. Acad. Sci. USA, 87: 2725-2729(1990); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992);Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwellet al., Science, 250: 1104-1110 (1990)).

Commonly used methods for detecting nucleic acid amplification productsrequire that the amplified product be separated from unreacted primers.This is typically achieved either through the use of gelelectrophoresis, which separates the amplification product from theprimers on the basis of a size differential, or through theimmobilization of the product, allowing free primer to be washed away.However, a number of methods for monitoring the amplification processwithout prior separation of primer have been described; all of them arebased on FET and none of them detect the amplified product directly.Instead, the methods detect some event related to amplification. Forthat reason, they are accompanied by problems of high background, andare not quantitative, as discussed below.

One method, described in Wang et al. (U.S. Pat. No. 5,348,853; and Anal.Chem., 67: 1197-1203 (1995)), uses an energy transfer system in whichenergy transfer occurs between two fluorophores on the probe. In thismethod, detection of the amplified molecule takes place in theamplification reaction vessel, without the need for a separation step.

A second method for detecting an amplification product without priorseparation of primer and product is the 5′-nuclease PCR assay (alsoreferred to as the TaqMan™ assay) (Holland et al., Proc. Natl. Acad.Sci. USA, 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res., 21:3761-3766 (1993)). This assay detects the accumulation of a specific PCRproduct by hybridization and cleavage of a doubly labeled fluorogenicprobe (the “TaqMan” probe) during the amplification reaction. Thefluorogenic probe consists of a nucleic acid labeled with both afluorescent reporter dye and a quencher dye. During PCR, this probe iscleaved by the 5′-exonuclease activity of DNA polymerase if, and onlyif, it hybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

Yet another method of detecting amplification products that relies onthe use of energy transfer is the “beacon probe” method described byTyagi et al. (Nature Biotech., 14: 303-309 (1996)) which is also thesubject of U.S. Pat. No. 5,312,728 to Lizardi et al. This method employsnucleic acid hybridization probes that can form hairpin structures. Onone end of the hybridization probe (either the 5′- or 3′-end) there is adonor fluorophore, and on the other end, an acceptor moiety. In thismethod, the acceptor moiety is a quencher, absorbing energy from thedonor. Thus when the beacon is in the open conformation, thefluorescence of the donor fluorophore is detectable, whereas when thebeacon is in hairpin (closed) conformation, the fluorescence of thedonor fluorophore is quenched. When employed in PCR, the molecularbeacon probe, which hybridizes to one of the strands of the PCR product,is in “open conformation,” and fluorescence is detected, while thosethat remain unhybridized will not fluoresce. As a result, the amount offluorescence will increase as the amount of PCR product increases, andthus can be used as a measure of the progress of the PCR.

The probes discussed above are most generally configured such that thequencher and fluorophore are on the 3′- and 5′-ends of the probe(Lyamichev et al., Science, 260:778-783 (1993)). This spacing of thefluorophore and quencher may impede fluorescent energy transfer:fluorescence energy transfer decreases as the inverse sixth power of thedistance between the fluorophore and quencher. Thus, if the quencher isnot close enough to the reporter to achieve efficient quenching thebackground emissions from the probe can be quite high.

To enable the coupling of a fluorescent label with a group ofcomplementary reactivity on a carrier molecule, a reactive derivative ofthe fluorophore is prepared. For example, Reedy et al. (U.S. Pat. No.6,331,632) describe cyanine dyes that are functionalized at anendocyclic nitrogen of a heteroaryl moiety with hydrocarbon linkerterminating in a hydroxyl moiety. The hydroxyl moiety is converted tothe corresponding phosphoramidite, providing a reagent for conjugatingthe cyanine dye to a nucleic acid. Waggoner (U.S. Pat. No. 5,627,027)has prepared derivatives of cyanine and related dyes that include areactive functional group through which the dye is conjugated to anotherspecies. The compounds set forth in Ohno et al. (U.S. Pat. No.5,106,990) include cyanine dyes that have a C₁-C₅ hydrocarbyl linkerterminated with a sulfonic acid, a carboxyl or a hydroxyl group. Randallet al. (U.S. Pat. Nos. 6,197,956; 6,114,350; 6,224,644; and 6,437,141)disclose cyanine dyes with a linker arm appended to an endocyclicheteroaryl nitrogen atom. The linkers include a thiol, amine or hydroxylgroup, or a protected analogue of these residues. Additional linkerarm-cyanine dyes are disclosed by Brush et al. (U.S. Pat. Nos.5,808,044; 5,986,086). These cyanine dyes are derivatized at bothendocyclic heteroaryl nitrogen atoms with a hydrocarbyl linkerterminating in a hydroxyl moiety. One hydroxyl moiety is converted tothe corresponding phoshporamidite and the other is protected as adimethoxytrityl ether.

None of the above-described references discloses or suggests modifyingthe fluorophore nucleus with a versatile amide-linked moiety that allowsfor the facile variation of the composition, length and degree ofbranching of the linker. Furthermore, none of the references suggests alinker that provides a locus for attaching the fluorophore to a solidsupport. Nor do the references describe a branched linker moiety thattethers both a phosphoramidite and dimethoxytrityl ether to a singleendocyclic nitrogen atom.

Attaching quenchers or fluorophores to sites other than the readilyaccessible 5′-OH group generally requires the synthesis of fluorescentlabels that attach the fluorophore to a single reactive residue of acarrier molecule or a selected reactive functional group on thatresidue; reacting the same fluorophore with a different functional groupof the carrier generally requires a new modification of the fluorescentcore. Similarly, modifying the structure or composition of the linkerarm requires a modification to the fluorophore nucleus. Thus, a cyaninelabel that provides a versatile entry point for an array of syntheticmodifications would represent a significant advance in the art.

BRIEF SUMMARY OF THE INVENTION

The inventors have prepared a class of cyanine-based fluorophoresmodified with a versatile linker arm, the structure of which is readilyalterable, thereby allowing the conjugation of the label to a variety ofpositions, through diverse functional groups, on a carrier molecule. Thecyanine-based labels are readily attached to a carrier molecule usingtechniques well known in the art, or modifications of such techniquesthat are well within the abilities of those of ordinary skill in theart. The versatility of the labels set forth herein provides a markedadvantage over currently utilized cyanine labels, probes assembled usingthose labels and methods relying upon such labels and probes. Moreover,the present invention provides a class of chemically versatile labels inwhich the fluorophore can be engineered to have a desired light emissionprofile.

Thus, in a first aspect, the present invention provides a fluorescentcompound having the formula:

in which A and B are independently selected from substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl moieties.Q is an unsaturated, substituted or unsubstituted, branched-, straight-or cyclic-alkyl or heteroalkyl moiety. Z is CH₂ or C(O).

The symbol R¹ represents a linker moiety, e.g., a substituted orunsubstituted alkyl, or a substituted or unsubstituted heteroalkylgroup. R¹ preferably does not comprise a carboxylate group. In anotherexemplary embodiment, R¹ does not include a moiety derived from acarboxylic acid group by replacement of the OH moiety, e.g., an ester,an amide, and an urethane.

In an exemplary embodiment, one or both of R² and R³ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl thatincludes a reactive functional group serving as a locus of attachmentbetween the fluorescent label and a carrier molecule, examples of whichinclude nucleic acids, amino acids, peptides, and saccharides. Inanother exemplary embodiment, one or both of R² and R³ are attached tothe carrier molecule through a residue derived from the reactive groupby its reaction with a complementary group on the carrier molecule. Oneof R² and R³ may also be H.

R¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl andoptionally includes a reactive group or a bond to a carrier molecule orsolid support.

In a second aspect, the invention provides a fluorescent compound havingthe formula:

in which R⁴-R¹¹ are generally species independently selected from H andother aryl group substituents as described herein, which optionallyinclude a reactive group. R¹ is a linker moiety, e.g., substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; andR¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl andoptionally includes a reactive group. Y¹ and Y² are membersindependently selected from (CR^(y1)R^(y2))_(v) in which each R^(y1) andR^(y2) is independently selected from H and those groups describedherein as substituents for alkyl moieties; and each index “v” isindependently selected from the integers 1 and 2.

Each L is an independently selected component of an unsaturated,substituted or unsubstituted, straight-, branched-, or cyclic-alkyl orheteroalkyl linker between the two heterocyclic moieties. An exemplarylinker component is C(R¹⁷), in which each R¹⁷ is independently selectedfrom H, and groups referred to herein as alkyl group substituents (e.g.,halide), substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl. The R¹⁷ groups on adjacent linker componentscan join up together to form a ring. The index “n” is an integer from 0to 4.

In a third aspect, the invention provides a fluorescent molecule havingthe formula:

in which the symbol R¹ represents substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups. R¹ preferably doesnot include a carboxylic acid group. In another exemplary embodiment, R¹does not include a moiety derived from a carboxylic acid group, e.g. anester, an amide, and an urethane.

R² is a group such as substituted or unsubstituted alkyl or substitutedor unsubstituted heteroalkyl. In an exemplary embodiment, R² includes amember selected from oxygen-containing reactive functional groups, andcarrier molecules, e.g., solid supports.

R³ represents a group such as H, substituted or unsubstituted alkyl, andsubstituted or unsubstituted heteroalkyl. R² and R³, together with thenitrogen to which they are attached, are optionally joined to form aring system. Exemplary ring systems include substituted or unsubstitutedC₅-C₇ cycloalkyl and substituted or unsubstituted 5-7-memberedheterocycloalkyl. R³ optionally includes a reactive group.

The symbols R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are each independentlyselected from groups such as substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, halogen, H, NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, andC(Z²)R²². Z¹ is either O or S. Z² represents O, S or NH. Groupscorresponding to R¹⁹ and R²⁰ are independently selected and include H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl. R²¹ is selected from H, substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl and C(Z³)R²². R²⁰ and R²¹,together with the nitrogen to which they are attached, can also be anynitrogen-containing reactive group. Exemplary groups include —NHNH₂,—N═C═S and —N═C═O.

R¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryland optionally includes a reactive group.

Z³ represents O, S or NH. The symbol R²² represents groups such assubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, OR²³, and NR²⁴R²⁵. R²³ represents H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²⁶. R²⁴ and R²⁵ are symbols representing groupsindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R²⁶ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl.

The symbols R¹³, R¹⁴, R¹⁵ and R¹⁶ represent groups that areindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl.

Each L is as discussed above.

The present invention also provides a conjugate between a carriermolecule, e.g., a nucleic acid, and a fluorescent compound of theinvention, which is covalently or ionically bound to a moiety of thecarrier molecule. When the carrier molecule is a nucleic acid,representative moieties at which the fluorescent compounds of theinvention are attached include the sugar moiety, at both O- andC-centers; endo- and exo-cyclic amines and carbon atoms of nucleobasemoieties, and internucleotide bridges. In still a further exemplaryembodiment, the conjugate between the compound of the invention and thecarrier molecule includes at least one moiety that quenches thefluorescence emission of the compound of the invention.

Other aspects, embodiments and objects of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D display representative compounds of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

“FET,” as used herein, refers to “Fluorescence Energy Transfer.”

“FRET,” as used herein, refers to “Fluorescence Resonance EnergyTransfer.” These terms are used herein to refer to both radiative andnon-radiative energy transfer processes. For example, processes in whicha photon is emitted and those involving long-range electron transfer areincluded within these terms. Throughout this specification, both ofthese phenomena are subsumed under the general term “donor-acceptorenergy transfer.”

Definitions

Where chemical moieties are specified by their conventional chemicalformulae, written from left to right, they equally encompass the moietywhich would result from writing the structure from right to left, e.g.,—CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is also intended torepresent. —S(O)₂HN—, etc.

“Cyanine,” as used herein, refers to polymethine dyes such as thosebased upon the cyanine, merocyanine, styryl and oxonol ring.

As used herein, “nucleic acid” means any natural or non-naturalnucleoside, or nucleotide and oligomers and polymers thereof, e.g., DNA,RNA, single-stranded, double-stranded, triple-stranded or more highlyaggregated hybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, conjugation with acompound of the invention or a construct that includes a compound of theinvention covalently attached to a linker that tethers the compound tothe nucleic acid, and those providing the nucleic acid with a group thatincorporates additional charge, polarizability, hydrogen bonding,electrostatic interaction, fluxionality or functionality to the nucleicacid. Exemplary modifications include the attachment to the nucleicacid, at any position, of one or more hydrophobic or hydrophilicmoieties, minor groove binders, intercalating agents, quenchers,chelating agents, metal chelates, solid supports, and other groups thatare usefully attached to nucleic acids.

Exemplary modified nucleic acids include, but are not limited to,peptide nucleic acids (PNAs), those with phosphodiester groupmodifications (e.g., replacement of O⁻ with OR, NR, or SR), 2′-, 3′- and5′-position sugar modifications, modifications to the base moiety, e.g.,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications, i.e.,substitution of P(O)O₃ with another moiety, methylations, unusualbase-pairing combinations such as the isobases, isocytidine andisoguanidine and the like. Nucleic acids can also include non-naturalbases, e.g., nitroindole. Non-natural bases include bases that aremodified with a compound of the invention or a linker-compound of theinvention construct, a minor groove binder, an intercalating agent, ahybridization enhancer, a chelating agent, a metal chelate, a quencher,a fluorophore, a fluorogenic compound, etc. Modifications within thescope of “nucleic acid” also include 3′ and 5′ modifications with one ormore of the species described above.

“Nucleic acid” also includes species that are modified at one or moreinternucleotide bridges (e.g., P(O)O₃) by replacing or derivatizing anoxygen of the bridge atom with a compound of the invention or a speciesthat includes a compound of the invention attached to a linker. Forexample, “nucleic acid” also refers to species in which the P(O)O₂moiety (the O⁻ moiety remains unchanged or is converted to “OR”) of anatural nucleic acid is replaced with a non-natural linker species,e.g., —ORP(O)O—, —ROP(O)R—, —ORP(O)OR—, —ROP(O)OR—, or —RP(O)R— in whichthe symbol “-” indicates the position of attachment of the linker to the2′-, 3′- or 5′-carbon of a nucleotide sugar moiety, thus allowing theplacement of the exemplified, and other, non-natural linkers betweenadjacent nucleoside sugar moieties. Exemplary linker subunits (“R”)include substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl moieties. “R” can include a compound of theinvention or a construct of a linker and a compound of the invention.

Furthermore, “nucleic acid” includes those species in which one or moreinternucleotide bridge does not include phosphorus: the bridge beingoptionally modified with a compound of the invention or a linkerarm-cyanine dye construct. An exemplary bridge includes a substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl moietyin which a carbon atom is the locus for the interconnection of twonucleoside sugar residues (or linker moieties attached thereto) and acompound of the invention or a linker construct that includes a compoundof the invention. The discussion above is not limited to moieties thatinclude a carbon atom as the point of attachment; the locus can also beanother appropriate linking atom, such as nitrogen or another atom.

Those of skill in the art will understand that in each of the “nucleicacid” compounds described above, the structure corresponding to the term“compound of the invention” can be interchanged with a quencher, ahybridization enhancer, and intercalator, a minor groove binder, achelating agent, a metal chelate or other moiety that is usefullyconjugated to a nucleic acid, optionally being present in tandem withspecies that include a compound of the invention or a derivativethereof.

As used herein, “quenching group” refers to any fluorescence-modifyinggroup of the invention that can attenuate at least partly the lightemitted by a fluorescent group. This attenuation is referred to hereinas “quenching”. Hence, illumination of the fluorescent group in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the fluorescent group and thequenching group.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. When the amino acids are α-amino acids, either theL-optical isomer or the D-optical isomer can be used. Additionally,unnatural amino acids, for example, β-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the D- orL-isomer. The L-isomers are generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. For a generalreview, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

“Bioactive species,” refers to molecules that, when administered to anorganism, affect that organism. Exemplary bioactive species includepharmaceuticals, pesticides, herbicides, growth regulators and the like.Bioactive species encompasses small molecules (i.e., approximately<1,000 daltons), oligomers, polymers and the like. Also included arenucleic acids and their analogues, peptides and their analogues and thelike.

“Carrier molecule,” as used herein refers to any molecule to which acompound of the invention is attached. Representative carrier moleculesinclude a protein (e.g., enzyme, antibody), glycoprotein, peptide,saccharide (e.g., mono-, oligo-, and poly-saccharides), hormone,receptor, antigen, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc., withoutlimitation. “Carrier molecule” also refers to species that might not beconsidered to fall within the classical definition of “a molecule,”e.g., solid support (e.g., synthesis support, chromatographic support,membrane), virus and microorganism.

“Activated derivatives of hydroxyl moieties,” and equivalent species,refer to compounds in which an oxygen-containing leaving group isformally accessed through a hydroxyl moiety.

“Activated derivatives of carboxyl moieties,” and equivalent species,refer to compounds in which an oxygen-containing leaving group isformally accessed through a carboxyl moiety.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturated alkylradicals include, but are not limited to, groups such as methyl,methylene, ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl,homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. An unsaturated alkyl group is one having one ormore double bonds or triple bonds. Examples of unsaturated alkyl groupsinclude, but are not limited to, vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, includes “alkylene”and those derivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups, which are limited to hydrocarbon groups,are termed “homoalkyl”.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. Accordingly,from the above discussion of substituents, one of skill in the art willunderstand that the terms “substituted alkyl” and “heteroalkyl” aremeant to include groups that have carbon atoms bound to groups otherthan hydrogen atoms, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

“Analyte”, “target”, “substance to be assayed”, and “target species,” asutilized herein refer to the species of interest in an assay mixture.The terms refer to a substance, which is detected qualitatively orquantitatively using a material, process or device of the presentinvention. Examples of such substances include cells and portionsthereof, enzymes, antibodies, antibody fragments and other biomolecules,e.g., antigens, polypeptides, glycoproteins, polysaccharides, complexglycolipids, nucleic acids, effector molecules, receptor molecules,enzymes, inhibitors and the like and drugs, pesticides, herbicides,agents of war and other bioactive agents.

More illustratively, such substances include, but are not limited to,tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), CA125, CA 19-9 and the like; various proteins, glycoproteins and complexglycolipids such as β₂-microglobulin (β₂ m), ferritin and the like;various hormones such as estradiol (E₂), estriol (E₃), human chorionicgonadotropin (hCG), luteinizing hormone (LH), human placental lactogen(hPL) and the like; various virus-related antigens and virus-relatedantibody molecules such as HBs antigen, anti-HBs antibody, HBc antigen,anti-HBc antibody, anti-HCV antibody, anti-HIV antibody and the like;various allergens and their corresponding IgE antibody molecules;narcotic drugs and medical drugs and metabolic products thereof; andnucleic acids having virus- and tumor-related polynucleotide sequences.

The term, “assay mixture,” refers to a mixture that includes the analyteand other components. The other components are, for example, diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like.

The term “drug” or “pharmaceutical agent,” refers to bioactive compoundsthat cause an effect in a biological organism. Drugs used as affinitymoieties or targets can be neutral or in their salt forms. Moreover, thecompounds can be used in the present method in a prodrug form. Prodrugsare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of interest in thepresent invention.

Introduction

The present invention provides a class of reactive fluorescent compoundsthat are based upon the cyanine nucleus. Also provided is a wide varietyof conjugates of the cyanine dyes with carrier molecules, includingbiological, non-biological and biologically active species. Selectedcyanine labels described herein include a functionalized linker arm thatis readily converted into an array of reactive derivatives withoutrequiring a modification of the cyanine nucleus. Accordingly, thecompounds of the invention provide an, as yet, undisclosed advantage,allowing facile access to an array of conjugates between the linkerarm-derivatized cyanine nucleus and carrier molecules.

Residing in the field of fluorescent labels, the present inventionprovides benefits of particular note. Fluorescent labels have theadvantage of requiring few precautions in handling, and being amenableto high-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Exemplary labels exhibit one or more of thefollowing characteristics: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling. Many fluorescent labels based upon the cyanine-nucleus arecommercially available from the SIGMA chemical company (Saint Louis,Mo.), Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis,Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., AldrichChemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersburg, Md.), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill. Furthermore, those of skill in the art will recognize howto select an appropriate cyanine-based fluorophore for a particularapplication and, if it not readily available commercially, will be ableto synthesize the necessary fluorophore de novo or synthetically modifycommercially available cyanine compounds to arrive at the desiredfluorescent label.

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

In a first aspect, the present invention provides a fluorescent compoundhaving the formula:

in which A and B are independently selected from substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl moieties.Q is an unsaturated, substituted or unsubstituted, branched-, straight-or cyclic-alkyl or heteroalkyl moiety. Z is CH₂ or C(O).

The symbol R¹ represents a linker moiety, e.g., a substituted orunsubstituted alkyl, or a substituted or unsubstituted heteroalkylgroup. R¹ preferably does not comprise a carboxylic acid group. Inanother exemplary embodiment, R¹ does not include a moiety derived froma carboxylic acid group by replacement of the OH moiety, e.g. an ester,an amide, and an urethane.

In an exemplary embodiment, one or both of R² and R³ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl thatincludes a reactive functional group serving as a locus of attachmentbetween the fluorescent label and a carrier molecule, examples of whichinclude nucleic acids, amino acids, peptides, and saccharides. Inanother exemplary embodiment, one or both of R² and R³ are attached tothe carrier molecule through a residue derived from the reactive groupby its reaction with a complementary group on the carrier molecule. Oneof R² and R³ may also be H.

In an exemplary embodiment, R² includes a member selected fromoxygen-containing reactive functional groups, solid supports and carriermolecules.

In another exemplary embodiment, R² and R³, together with the nitrogento which they are attached, are optionally joined to form a ring system.Exemplary ring systems include substituted or unsubstituted C₅-C₇cycloalkyl and substituted or unsubstituted 5-7-memberedheterocycloalkyl.

R¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl andoptionally includes a reactive group or a bond to a carrier molecule orsolid support.

In a second aspect, the invention provides a fluorescent compound havingthe formula:

in which R⁴-R¹¹ are generally species independently selected from H andother aryl group substituents as described herein, which optionallyinclude a reactive group. R¹ is a linker moiety, e.g., substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; andR¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl andoptionally includes a reactive group. Y¹ and Y² are membersindependently selected from (CR^(y1)R^(y2))_(v) in which each R^(y1) andR^(y2) is independently selected from H and those groups describedherein as substituents for alkyl moieties; and each index “v” isindependently selected from the integers 1 and 2.

Each L is an independently selected component of an unsaturated,substituted or unsubstituted, straight-, branched-, or cyclic-alkyl orheteroalkyl linker between the two heterocyclic moieties. An exemplarylinker component is C(R¹⁷), in which each R¹⁷ is independently selectedfrom H, and groups referred to herein as alkyl group substituents (e.g.,halide), substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl. The R¹⁷ groups on adjacent linker componentscan join up together to form a ring. The index “n” is an integer from 0to 4.

In a third aspect, the invention provides a fluorescent molecule havingthe formula:

in which the symbol R¹ represents substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups. R¹ preferably doesnot include a carboxylic acid group. In another exemplary embodiment, R¹does not include a moiety derived from a carboxylic acid group, e.g. anester, an amide, and an urethane.

R² is a group such as substituted or unsubstituted alkyl or substitutedor unsubstituted heteroalkyl. In an exemplary embodiment, R² includes amember selected from oxygen-containing reactive functional groups, andcarrier molecules, e.g., solid supports.

R³ represents a group such as H, substituted or unsubstituted alkyl, andsubstituted or unsubstituted heteroalkyl. R² and R³, together with thenitrogen to which they are attached, are optionally joined to form aring system. Exemplary ring systems include substituted or unsubstitutedC₅-C₇ cycloalkyl and substituted or unsubstituted 5-7-memberedheterocycloalkyl. R³ optionally includes a reactive group.

The symbols R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are each independentlyselected from groups such as substituted or unsubstituted alkyl,substituted or unsubstituted heteroralkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, halogen, H, NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, andC(Z²)R²². Z¹ is either O or S. Z² represents O, S or NH. Groupscorresponding to R¹⁹ and R²⁰ are independently selected and include H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl. R²¹ is selected from H, substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl and C(Z³)R²². R²⁰ and R²¹,together with the nitrogen to which they are attached, can also be anynitrogen-containing reactive group. Exemplary groups include —NHNH₂,—N═C═S and —N═C═O.

R¹² is substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryland optionally includes a reactive group.

Z³ represents O, S or NH. The symbol R²² represents groups such assubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, OR²³, and NR²⁴R²⁵. R²³ represents H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, R²⁴substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²⁶. R²⁴ and R²⁵ are symbols representing groupsindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R²⁶ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl.

The symbols R¹³, R¹⁴, R¹⁵ and R¹⁶ represent groups that areindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl.

Each L is as discussed above.

Representative -(L=L)_(n)-L=moieties of use in the various aspects ofthe invention include:

in which Z⁴ is H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl or another moiety selected from the substituents foralkyl moieties described herein

In a representative embodiment, the invention provides a cyanine dye inwhich R² includes a moiety having the formula:

wherein L¹, L² and L³ are members independently selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The index “t” is 0 or 1.

A subset of R² moieties according to the motif set forth above have theformula:

in which the symbols R^(x) and R^(y) represent groups that areindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl, a hydroxyl-protecting group, aphosphate moiety, a phosphodiester moiety, a phosphorus-containinginternucleotide bridge, a solid support, a carrier molecule and—OP(OR^(o))(N(R^(p)R^(q))). The groups represented by the symbols R^(o),R^(P) and R^(q) are members independently selected from H, substitutedor unsubstituted C₁-C₆ alkyl and substituted or unsubstituted C₁-C₆heteroalkyl; and the index “s” is an integer from 1 to 20. In anexemplary embodiment, R^(o) is CH₂CH₂CN.

The invention also provides fluorescent compounds in which at least oneof R^(x) and R^(y) comprises a moiety having the formula:

L⁴ is a member selected from a bond, substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl; and R^(z) is a memberselected from a reactive functional group, solid support, a nucleicacid, a saccharide and a peptide. In selected compounds of theinvention, L⁴ comprises a moiety having the formula:

wherein the symbol Z³ represents either CH₂ or C═O.

In another embodiment, the invention provides cyanine dyes in which oneof the substituents on the cyanine nucleus, preferably R¹², includes amoiety having the structure:

in which L^(1a) is a member selected from substituted or unsubstitutedalkyl, and substituted or unsubstituted heteroalkyl groups. The symbolsR^(2a) and R^(3a) represent groups that are independently selected fromH, substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl. The groups R² and R³, together with the nitrogen to whichthey are attached, are optionally joined to form a ring. Preferred ringstructures include substituted or unsubstituted C₅-C₇ cycloalkyl andsubstituted or unsubstituted 5-7-membered heterocycloalkyl.

An exemplary linker species according to the motif presented aboveincludes an NR²R³ moiety that has the formula:

in which R^(2a) and R^(3a) are members independently selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The symbols X^(2a) and X^(3a) represent groups that areindependently selected from H, substituted or unsubstituted lower alkyl,substituted or unsubstituted heteroalkyl, reactive functional groups anda bond to a carrier molecule. When the carrier molecule is a nucleicacid, the bond can be to a nucleobase (e.g., to C or N), sugar (e.g., toC or O) or internucleotide bridge (e.g., to P, O, S, C or N).

Exemplary identities for X^(2a) and X^(3a) include —CH₃, —OH, —COOH,—NH₂, —SH, and —OP(OX′)(N(X″)₂) in which X′ and X″ are membersindependently selected from substituted or unsubstituted alkyl. In anexemplary compound of the invention X′ is cyanoethyl; and both X″moieties are isopropyl. When X^(2a) and X^(3a) are components oflinkages between a species of the invention and a carrier molecule, thegroup is modified in a manner that satisfies the rules of valence, e.g.,—OH becomes —O—; COOH becomes COOR, CONRR′, etc.

In another preferred embodiment, a member selected from R^(2a), R^(3a)and combinations thereof comprises a polyether. Preferred polyethersinclude, for example, poly(ethylene glycol), poly(propyleneglycol) andcopolymers thereof. The polyether may be internal to R^(2a) or R^(3a)group or it may form the free terminus of the group. When the polyetheris at the terminus of the group, the terminal —O— moiety is present as—OH, alkoxy or one of a variety of the groups referred to herein assubstituents for alkyl moieties. See, for example, Shearwater Polymers,Inc., Catalog: Polyethylene Glycol Derivatives 2002.

In a further exemplary embodiment, NR²R³ has the formula:

in which the indexes p and q are integers independently selected from 1to 20, inclusive, preferably from 2 to 16, inclusive.

In yet another exemplary embodiment, NR²R³ has the formula:

in which the index “v” is 0 or 1. R^(2a) and R^(3a) are independentlyselected substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl moieties. When “v” is 0, the phosphate showncan alternatively be OH. Although represented as interposed between twonucleotides, the fluorescent label of the invention can be placed at anypoint between two nucleoside or nucleotide subunits in a nucleic acid.Thus, exemplary compounds include NR²R³ at an internal position of thenucleic acid, and tethered to the nucleic acid at the linkage betweenthe 5′ and 5′-1 residues and/or the linkage between the 3′ and 3′-1residues

In a further exemplary embodiment, NR²R³ has the formula:

When “v” is 0, the phosphate group is optionally an OH group.

The invention also provides nucleic acid derivatives in which a compoundof the invention is conjugated to a sugar moiety of the nucleic acid. Anexemplary species according to this motif has the formula:

in which the index u represents 0, 1 or a number greater than one.Although shown attached to the 2′carbon of the 3′-terminus of thenucleic acid, those of skill will appreciate that a similar structuretethered to the 5′-terminus, or an internal site of the nucleic acid iswithin the scope of the invention. Moreover, the group can be tetheredthrough the O atom of a 2′-hydroxyl. When “u” is 0, thephosphate/phosphodiester group is optionally OH.

Moreover, the agents of the invention can be conjugated through the 3′-and/or 5′-hydroxyl moiety of a nucleic acid.

Representative compounds of the invention are set forth in FIG. 1.

In another exemplary embodiment, the invention provides cyanine dyeshaving a formula selected from:

in which the symbol R³⁰ represents OH, OP(OR^(o))(NR^(p)R^(q)))₂,-L^(c)-R³¹ or R³⁵O(CH₂)_(j)L^(c)(R³¹), in which (CH₂)_(c) is attached toL^(c). L^(c) is a bond or linker selected from substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl bondedto R³¹. R³¹ is OH, OP(OR^(o))(N(R^(p)R^(q))), a nucleic acid, aphosphoramidite of a nucleic acid, a nucleic acid linked to a solidsupport, and amino acid, a protected amino acid, an amino acid attachedto a solid support, an amino acid residue of a peptide, a carboxylate oran activated carboxylate. The symbols R^(o), R^(p) and R^(q)independently represent H, substituted or unsubstituted C₁-C₆ alkyl orsubstituted or unsubstituted C₁-C₆ heteroalkyl. The index c is aninteger from 0 to 20, and the index j is an integer selected from 1 to20. R³⁵ is H or a hydroxyl protecting group (e.g., trityl or substitutedtrityl). R¹-R¹⁶ are as discussed above.

An exemplary species according to L^(c) has the formula:

in which the indices d and s are independently selected from theintegers from 0 to 10. The index e is an integer from 0 to 1,000.Generally, at least one of d, s and e is at least 1. A is a bond, NH, Sor O. The symbol R³¹ is as discussed above.

In another exemplary embodiment, -L^(c)-R³¹ has the formula:

wherein the symbol A represents O, S or NH. B is a nucleic acid base.R³² is H, P(OR^(o))(N(R^(p)R^(q))) or L^(g)-R³⁴. L^(g) is a bond, aninternucleotide phosphodiester bridge, a substituted or unsubstitutedalkyl group or a substituted or unsubstituted heteroalkyl moiety. Thesymbol R³⁴ represents OH, a solid support, P(OR^(o))(N(R^(p)R^(q))) or anucleic acid.

R³³ is a hydroxyl protecting group (e.g., trityl or a substitutedtrityl, see, for example, Jones, AMINO ACID AND PEPTIDE SYNTHESIS,Oxford Science Publications, Oxford (1992)), OH, a solid support,P(OR^(o))(N(R^(p)R^(q))), or a nucleic acid.

An exemplary nucleic acid according to this embodiment has the formula:

in which R³² and R³³ are as discussed above.

In another embodiment, the cyanine fluorophore is attached to an aminoacid, preferably through a linker. For example compounds within thescope of the present invention include those in which R¹² has theformula:

in which J is H or an amine protecting group, generally a protectinggroup recognized in the peptide synthesis art (e.g., t-Boc, FMOC, etc.;see, for example, Jones, AMINO ACID AND PEPTIDE SYNTHESIS, OxfordScience Publications, Oxford (1992)). J¹ is a member selected from H,activating groups and amino acids. J² is H, substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl. A and the indices d,s and e are as discussed above.

In yet a further exemplary embodiment, the invention provides a compoundin which R³² has the formula:

wherein A¹ and A² are independently selected from a bond, O and NH. Thesymbol SS represents a solid support. Q is a member selected from O⁻ andsubstituted or unsubstituted alkyl; and f, g, and h are integersindependently selected from 0 to 20. In selected species according tothis motif A¹ and A² are both O.Synthesis

The compounds of the invention are synthesized by an appropriatecombination of generally well-known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention. It is not intendedto define the scope of reactions or reaction sequences that are usefulin preparing the compounds of the present invention.

The compounds of the invention can be prepared as a single isomer or amixture of isomers, including, for example cis-isomers, trans-isomers,diastereomers and stereoisomers. In a preferred embodiment, thecompounds are prepared as substantially a single isomer. Isomericallypure compounds are prepared by using synthetic intermediates that areisomerically pure in combination with reactions that either leave thestereochemistry at a chiral center unchanged or result in its completeinversion. Alternatively, the final product or intermediates along thesynthetic route can be resolved into a single isomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate resolution or synthetic method for a particular situation.See, generally, Furniss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICALORGANIC CHEMISTRY 5^(TH) ED., Longman Scientific an d Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

An exemplary synthetic route is set forth in Scheme 1.(1-(ε-Carboxypentyl)-1′-(ethyl)-indo)-carbocyanine dye 1 is prepared bycoupling 1-(ε-carboxypentyl)-2,3,3-trimethylindoleninium and1-ethyl-2,3,3-trimethylindoleninium with N,N′-diphenylformamidine. TheN-hydroxysucinimide ester of the dye 2 is prepared by the DCC mediatedcoupling of N-hydroxysuccinimide with 1.

Scheme 2 outlines the preparation of a serinol linker 3 and itsconjugation to dye 1. Thus, the amine moiety of serinol is protected asthe trifluoroacetic acid amide, one of the primary hydroxyl groups isconverted to the dimethoxytrityl ether and the TFA moiety issubsequently removed by hydrolysis with KOH. The serinol linker 3 iscoupled to dye 1 to produce conjugate 4, using Bop andN-methylmorpholine. The free hydroxyl of the conjugate is converted tothe corresponding phosphoramidite 10 with phosphane and 1-H-tetrazole.The phosphoramidite is utilized in the solid phase synthesis of 15-meroligonucleotide 11.

Scheme 3 sets out an exemplary route for preparing a linker armderivatized dye and its attachment to a solid support. The serinol dyeconjugate 4 was acylated with diglycolic anhydride, forming 5. A solidsupport with a dye of the invention immobilized thereon was prepared bycombining 5 with aminopropylated controlled pore glass in the presenceof Bop and HOBT.

As shown in Scheme 4, compound 1 is a versatile intermediate, allowingaccess to a variety of linker derivatized cyanine dyes. Thus, 1 isconverted to the corresponding 2-(2-aminoethoxy)-ethanol derivative bycoupling this moiety to the carboxylic acid moiety of the dye to form 7.The hydroxyl moiety of 7 is converted to a phosphoramidite, affording 8,which is utilized in the solid phase synthesis of 15-mer oligonucleotide9.

Linkers of use in the invention also include cyclic structures. As shownin Scheme 5, N-hydroxypiperidine is conjugated to 1, forming 12, whichis converted to phosphoramidate 13. The phosphoramidate is utilized inthe solid phase synthesis of dye conjugated 15-mer oligonucleotide 14.

Another amino-alcohol of use as a linker is 6-amino-1-hexanol, which, asshown in Scheme 6, is coupled to dye 1, forming conjugate 15. Thehydroxyl moiety of 15 is converted to the phosphoramidate, providing 16,which is utilized in the preparation of dye conjugated 15-meroligonucleotide 17.

Chemical synthesis of the nucleic acid is generally automated and isperformed by coupling nucleosides through phosphorus-containing covalentlinkages. The most commonly used oligonucleotide synthesis methodinvolved reacting a nucleoside with a protected cyanoethylphosphoramidite monomer in the presence of a weak acid. The couplingstep is followed by oxidation of the resulting phosphite linkage.Finally, the cyanoethyl protecting group is removed and the nucleic acidis cleaved from the solid support on which it was synthesized. Thelabels of the present invention can be incorporated duringoligonucleotide synthesis using a mono- or bis-phosphoramiditederivative of the fluorescent compound of the invention. Alternatively,the label can be introduced by combining a compound of the inventionthat includes a reactive functional group with the nucleic acid underappropriate conditions to couple the compound to the nucleic acid. Inyet another embodiment, the fluorescent compound is attached to a solidsupport through a linker arm, such as a substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl or a nucleic acidresidue. Synthesis proceeds with the fluorescent moiety already in placeon the growing nucleic acid chain.

Enzymatic methods of synthesis involve the use of fluorescent-labelednucleic acids in conjunction with a nucleic acid template, a primer andan enzyme. Efficient enzymatic incorporation of a fluorescent-labelednucleic acid is facilitated by selection of reaction partners that donot adversely affect the enzymes ability to couple the partners.

In those embodiments of the invention in which the cyanine-basedfluorescent compound of the invention is attached to a nucleic acid, thecarrier molecule is produced by either synthetic (solid phase, liquidphase or a combination) or enzymatically or by a combination of theseprocesses.

Another synthetic strategy for the preparation of oligonucleotides isthe H-phosphonate method (B. Froehler and M. Matteucci, TetrahedronLett., vol 27, p 469-472, 1986). This method utilizes activatednucleoside H-phosphonate monomers rather than phosphoramidites to createthe phosphate internucleotide linkage. In contrast to thephosphoramidite method, the resulting phosphonate linkage does notrequire oxidation every cycle but instead only a single oxidation stepat the end of chain assembly. The H-phosphonate method may also be usedto conjugate reporters and dyes to synthetic oligonucleotide chains (N.Sinha and R. Cook, Nucleic Acids Research, Vol 16, p. 2659, 1988).

Reactive Functional Groups

The compounds of the invention bear a reactive functional group, whichcan be located at any position on the molecule. Exemplary speciesinclude a reactive functional group as a constituent of at least one ofR² and R³. When the reactive group is attached a substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl moiety,the reactive group is preferably located at a terminal position of thealkyl or heteroalkyl chain. Reactive groups and classes of reactionsuseful in practicing the present invention are generally those that arewell known in the art of bioconjugate chemistry. Currently favoredclasses of reactions available with reactive cyanine-based compounds ofthe invention are those proceeding under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction, Diels-Alder addition). These and other useful reactions arediscussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed.,John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES,Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OFPROTEINS; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

Useful reactive functional groups include, for example:

-   -   (a) carboxyl groups and derivatives thereof including, but not        limited to activated esters, e.g., N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters, activating groups used in peptide synthesis and        acid halides;    -   (b) hydroxyl groups, which can be converted to esters,        sulfonates, phosphoramidates, ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be displaced with a        nucleophilic group such as, for example, an amine, a carboxylate        anion, thiol anion, carbanion, or an alkoxide ion, thereby        resulting in the covalent attachment of a new group at the site        of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, allowing derivatization via        formation of carbonyl derivatives, e.g., imines, hydrazones,        semicarbazones or oximes, or via such mechanisms as Grignard        addition or alkyllithium addition;    -   (f) sulfonyl halide groups for reaction with amines, for        example, to form sulfonamides;    -   (g) thiol groups, which can be converted to disulfides or        reacted with acyl halides, for example;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assembleor utilize the reactive cyanine analogue. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In addition to those embodiments in which a compound of the invention isattached directly to a carrier molecule, the fluorophores can also beattached by indirect means. In this embodiment, a ligand molecule (e.g.,biotin) is generally covalently bound to the probe species. The ligandthen binds to another molecules (e.g., streptavidin) molecule, which iseither inherently detectable or covalently bound to a signal system,such as a fluorescent compound, or an enzyme that produces a fluorescentcompound by conversion of a non-fluorescent compound. Useful enzymes ofinterest as labels include, for example, hydrolases, particularlyphosphatases, esterases and glycosidases, hydrolases, peptidases oroxidases, and peroxidases.

Probes

The invention provides probes having a cyanine dye of the inventionconjugated to a carrier molecule, for example, a target species (e.g.,receptor, enzyme, etc.) a ligand for a target species (e.g., nucleicacid, peptide, etc.), a small molecule (e.g., drug, pesticide, etc.), asolid support and the like. The probes can be used for in vitro and invivo applications.

An unexpected property of the cyanine dye of the invention is theirrobustness under a variety of synthetic conditions used to attach thecyanine dye of the invention to a carrier molecule. For example, many ofthe cyanine dyes of the invention survive the conditions necessary forautomated synthesis of nucleic acids without undergoing any substantialdegree of degradation or alteration. In contrast, many of art-recognizedfluorophores presently in use require the use of special conditions toassemble the carrier molecule to which they are attached, or they haveto be attached after the completion of the carrier molecule synthesis.The additional complexity of the synthesis of a probe increases both theduration of the synthesis and its cost.

Small Molecule Probes

The cyanine dyes of the invention can be used as components of smallmolecule probes. In a preferred design, a small molecule probe includesa cyanine dye of the invention and a second species that alters theluminescent properties of the dyes, e.g., a quencher of fluorescence. Inan exemplary embodiment, an agent, such as an enzyme cleaves the cyaninedye of the invention, the quencher or both from the small moleculegenerating fluorescence in the system under investigation (see, forexample, Zlokarnik et al., Science 279: 84-88 (1998)).

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a cyanine dyeof the invention is used as a capture probe. The nucleic acid probe canbe used in solution phase or it can be attached to a solid support. Theimmobilized probes can be attached directly to the solid support orthrough a linker arm between the support and the cyanine dye or betweenthe support and a nucleic acid residue. Preferably, the probe isattached to the solid support by a linker (i.e., spacer arm, supra). Thelinker serves to distance the probe from the solid support. The linkeris most preferably from about 5 to about 30 atoms in length, morepreferably from about 10 to about 50 atoms in length. Exemplaryattachment points include the 3′- or 5′-terminal nucleotide of the probeas well as other accessible sites discussed herein.

In yet another preferred embodiment, the solid support is also used asthe synthesis support in preparing the probe. The length and chemicalstability of the linker between the solid support and the first 3′-unitof nucleic acid (or the cyanine dye) play an important role in efficientsynthesis and hybridization of support bound nucleic acids. The linkerarm should be sufficiently long so that a high yield (>97%) can beachieved during automated synthesis. The required length of the linkerwill depend on the particular solid support used. Exemplary linker arefrom about 6 to about 30 atoms in length. For nucleic acid synthesis,the linker arm is usually attached to the 3′-OH of the 3′-terminus by acleaveable linkage, e.g., an ester linkage, which can be cleaved withappropriate reagents to free the nucleic acid from the solid support.

Hybridization of a probe immobilized on a solid support generallyrequires that the probe be separated from the solid support. A preferredlinker for this embodiment includes at least about 20 atoms, morepreferably at least about 50 atoms.

A wide variety of linkers are known in the art, which may be used toattach the nucleic acid probe to the solid support. The linker may beformed of any moiety or combination of moieties, which does notsignificantly interfere with the hybridization of the target sequence tothe probe attached to the solid support. The linker may be formed of,for example, a homopolymeric nucleic acid, which can be readily added onto the linker by automated synthesis. Alternatively, polymers such aspolyethylene glycol can be used as the linker. Such polymers arepresently preferred over homopolymeric nucleic acids because they do notsignificantly interfere with the hybridization of probe to the targetnucleic acid. Polyethylene glycol is particularly preferred because itis commercially available, soluble in both organic and aqueous media,easy to functionalize, and completely stable under nucleic acidsynthesis and post-synthesis conditions.

The linkages between the solid support, the linker and the probe arepreferably not cleaved during synthesis or removal of base protectinggroups under basic conditions at high temperature. These linkages can,however, be selected from groups that are cleavable under a variety ofconditions. Examples of presently preferred linkages include carbamate,ester and amide linkages.

Dual Labeled Probes

The present invention also provides dual labeled probes that includeboth a cyanine dye of the invention and another label. Exemplary duallabeled probes include nucleic acid probes that include a nucleic acidwith a cyanine dye of the invention attached thereto. Exemplary probesinclude both a cyanine dye of the invention and a quencher. The probesare of use in a variety of assay formats. For example, when a nucleicacid singly labeled with a cyanine dye of the invention is the probe,the interaction between the first and second nucleic acids can bedetected by observing the interaction between the cyanine dye of theinvention and the nucleic acid. Alternatively, the interaction is thequenching by a quencher attached to the second nucleic acid of thefluorescence from a cyanine dye of the invention.

The cyanine dyes of the invention are useful in conjunction withnucleic-acid probes in a variety of nucleic acidamplification/quantification strategies including, for example,5′-nuclease assay, Strand Displacement Amplification (SDA), Nucleic AcidSequence-Based Amplification (NASBA), Rolling Circle Amplification(RCA), as well as for direct detection of targets in solution phase orsolid phase (e.g., array) assays. Furthermore, the cyanine dye of theinvention-derivatized nucleic acids can be used in probes ofsubstantially any format, including, for example, format selected frommolecular beacons, Scorpion Probes™, Sunrise Probes™, conformationallyassisted probes, light up probes, Invader Detection probes, and TaqMan™probes. See, for example, Cardullo, R., et al., Proc. Natl. Acad. Sci.USA, 85:8790-8794 (1988); Dexter, D. L., J. Chem. Physics, 21:836-850(1953); Hochstrasser, R. A., et al., Biophysical Chemistry, 45:133-141(1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995);Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann.Rev. Biochem., 47:819-846 (1978); Wang, G., et al., Tetrahedron Letters,31:6493-6496 (1990); Wang, Y., et al., Anal. Chem., 67:1197-1203 (1995);Debouck, C., et al., in supplement to nature genetics, 21:48-50 (1999);Rehman, F. N., et al., Nucleic Acids Research, 27:649-655 (1999);Cooper, J. P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, E. M.,et al., Genome Methods, 6:995-1001 (1996); Hochstrasser, R. A., et al.,Biophysical Chemistry, 45:133-141 (1992); Holland, P. M., et al., ProcNatl. Acad. Sci. USA, 88:7276-7289 (1991); Lee, L. G., et al., NucleicAcids Rsch., 21:3761-3766 (1993); Livak, K. J., et al., PCR Methods andApplications, Cold Spring Harbor Press (1995); Vamosi, G., et al.,Biophysical Journal, 71:972-994 (1996); Wittwer, C. T., et al.,Biotechniques, 22:176-181 (1997); Wittwer, C. T., et al., Biotechniques,22:130-38 (1997); Giesendorf, B. A. J., et al., Clinical Chemistry,44:482-486 (1998); Kostrikis, L. G., et al., Science, 279:1228-1229(1998); Matsuo, T., Biochemica et Biophysica Acta, 1379:178-184 (1998);Piatek, A. S., et al., Nature Biotechnology, 16:359-363 (1998);Schofield, P., et al., Appl. Environ. Microbiology, 63:1143-1147 (1997);Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., etal., Nature Biotechnology, 14:303-308 (1996); Nazarenko, I. A., et al.,Nucleic Acids Research, 25:2516-2521 (1997); Uehara, H., et al.,Biotechniques, 26:552-558 (1999); D. Whitcombe, et al., NatureBiotechnology, 17:804-807 (1999); Lyamichev, V., et al., NatureBiotechnology, 17:292 (1999); Daubendiek, et al., Nature Biotechnology,15:273-277 (1997); Lizardi, P. M., et al., Nature Genetics, 19:225-232(1998); Walker, G., et al., Nucleic Acids Res., 20:1691-1696 (1992);Walker, G. T., et al., Clinical Chemistry, 42:9-13 (1996); and Compton,J., Nature, 350:91-92 (1991).

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art.

More specifically, there are many linking moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink TM II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Exemplary fluorophores that can be combined in a probe with a cyaninedye of the invention include those set forth in Table 1.

TABLE 1 Suitable moieties that can be selected as donors or acceptors indonor-acceptor energy transfer pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives:   acridine   acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin   7-amino-4-methylcoumarin (AMC,Coumarin 120)   7-amino-4-trifluoromethylcouluarin (Coumaran 151)cyanine dyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives:   eosin   eosin isothiocyanate erythrosin and derivatives:  erythrosin B   erythrosin isothiocyanate ethidium fluorescein andderivatives:   5-carboxyfluorescein (FAM)  5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)  2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)   fluorescein  fluorescein isothiocyanate   QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives:   pyrene   pyrene butyrate  succinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives:   6-carboxy-X-rhodamine(ROX)   6-carboxyrhodamine (R6G)   lissamine rhodamine B sulfonylchloride rhodamine (Rhod)   rhodamine B   rhodamine 123   rhodamine Xisothiocyanate   sulforhodamine B   sulforhodamine 101 sulfonyl chloridederivative of sulforhodamine 101 (Texas Red)N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine  tetramethyl rhodamine isothiocyanate (TRITC) riboflavin rosolic acidterbium chelate derivatives

There is a great deal of practical guidance available in the literaturefor selecting appropriate donor-acceptor pairs for particular probes, asexemplified by the following references: Pesce et al., Eds.,FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al.,FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing reporter-quencher pairs (see,for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATICMOLECULES, 2nd Edition (Academic Press, New York, 1971); Griffiths,COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York,1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

As will be apparent to those of skill in the art the methods set forthabove are equally applicable to the coupling to a nucleic acid of groupsother than the fluorescent compounds of the invention, e.g., quenchers,intercalating agents, hybridization enhancing moieties, minor groovebinders, alkylating agents, cleaving agents, etc.

For example, in selected embodiments, the probe includes a metal chelateor a chelating agent attached to the carrier molecule. The use of thesecompounds to bind to specific compounds is well known to those of skillin the art. See, for example, Pitt et al. “The Design of ChelatingAgents for the Treatment of Iron Overload,” In, INORGANIC CHEMISTRY INBIOLOGY AND MEDICINE; Martell, A. E., Ed.; American Chemical Society,Washington, D.C., 1980, pp. 279-312; Lindoy, L. F., THE CHEMISTRY OFMACROCYCLIC LIGAND COMPLEXES; Cambridge University Press, Cambridge,1989; Dugas, H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989,and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, R. E., Whitaker, J. R., Eds., American ChemicalSociety, Washington, D.C., 1982, pp. 370-387; Kasina et al. BioconjugateChem. 9:108-117 (1998); Song et al., Bioconjugate Chem. 8:249-255(1997).

In a presently preferred embodiment, the chelating agent is apolyaminocarboxylate chelating agent such as ethylenediaminetetraaceticacid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). Thesechelating agents can be attached to any amine-terminated component of acarrier molecule or a spacer arm, for example, by utilizing thecommercially available dianhydride (Aldrich Chemical Co., Milwaukee,Wis.).

The nucleic acids for use in the probes of the invention can be anysuitable size, and are preferably in the range of from about 10 to about100 nucleotides, more preferably from about 10 to about 80 nucleotidesand more preferably still, from about 20 to about 40 nucleotides. Theprecise sequence and length of a nucleic acid probe of the inventiondepends in part on the nature of the target polynucleotide to which itbinds. The binding location and length may be varied to achieveappropriate annealing and melting properties for a particularembodiment. Guidance for making such design choices can be found in manyart-recognized references.

Preferably, the 3′-terminal nucleotide of the nucleic acid probe isblocked or rendered incapable of extension by a nucleic acid polymerase.Such blocking is conveniently carried out by the attachment of a donoror acceptor moiety to the terminal 3′-position of the nucleic acidprobe, either directly or by a linking moiety.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, as discussed above, the nucleic acid can be modified at thebase moiety, sugar moiety, or phosphate backbone with other groups suchas radioactive labels, minor groove binders, intercalating agents, donorand/or acceptor moieties and the like.

For example, the nucleic acid can comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,nitroindole, and 2,6-diaminopurine. The cyanine dye of the invention oranother probe component can be attached to the modified base.

In another embodiment, the nucleic acid comprises at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose. The cyanine dye oranother probe component can be attached to the modified sugar moiety.

In yet another embodiment, the nucleic acid comprises at least onemodified phosphate backbone selected from the group including, but notlimited to, a peptide nucleic acid hybrid, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof. The cyanine dye or another probe componentcan be attached to the modified phosphate backbone.

Phosphodiester linked nucleic acids of the invention can be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer using commercially available amidite chemistries (Ozaki etal., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al.,Nucleic Acids Research, 18: 5419-5423 (1990); Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679). Nucleic acidsbearing modified phosphodiester linking groups can be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids can be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

When the nucleic acids are synthesized utilizing an automated nucleicacid synthesizer, the donor and acceptor moieties are preferablyintroduced during automated synthesis. Alternatively, one or more ofthese moieties can be introduced either before or after the automatedsynthesis procedure has commenced. For example, donor and/or acceptorgroups can be introduced at the 3′-terminus using a solid supportmodified with the desired group(s). Additionally, donor and/or acceptorgroups can be introduced at the 5′-terminus by, for example a derivativeof the group that includes a phosphoramidite. In another exemplaryembodiment, one or more of the donor and/or acceptor groups isintroduced after the automated synthesis is complete.

In the dual labeled probes, the quencher moiety is preferably separatedfrom the cyanine dye of the invention by at least about 10 nucleotides,and more preferably by at least about 15 nucleotides. The quenchermoiety is preferably attached to either the 3′- or 5′-terminalnucleotides of the probe. The cyanine dye of the invention moiety isalso preferably attached to either the 3′- or 5′-terminal nucleotides ofthe probe. More preferably, the donor and acceptor moieties are attachedto the 3′- and 5′- or 5′- and 3′-terminal nucleotides of the probe,respectively, although internal placement is also useful.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g.,butylamine:water 1:3, 8 hours, 70° C.). Deprotection under theseconditions is facilitated by the use of quick deprotect amidites (e.g.,dC-acetyl, dG-dmf).

Following cleavage from the support and deprotection, the nucleic acidis purified by any method known in the art, including chromatography,extraction and gel purification. In a preferred embodiment, the nucleicacid is purified using HPLC. The concentration and purity of theisolated nucleic acid is preferably determined by measuring the opticaldensity at 260 nm in a spectrophotometer.

Peptide Probes

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a cyanine dye of the invention can be used in both in vivoand in vitro enzymatic assays.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a cyanine dye of theinvention; and iii) a cleavage or assembly recognition site for theenzyme. Moreover, the peptide construct is preferably exists in at leastone conformation that allows donor-acceptor energy transfer between thecyanine dye of the invention and the quencher when the fluorophore isexcited.

In the dual labeled probes of the invention, the donor and acceptormoieties are connected through an intervening linker moiety. The linkermoiety, preferably, includes a peptide moiety, but can be or can includeanother organic molecular moiety, as well. In a preferred embodiment,the linker moiety includes a cleavage recognition site specific for anenzyme or other cleavage agent of interest. A cleavage site in thelinker moiety is useful because when a tandem construct is mixed withthe cleavage agent, the linker is a substrate for cleavage by thecleavage agent. Rupture of the linker moiety results in separation ofthe cyanine dye and the quencher. The separation is measurable as achange in donor-acceptor energy transfer. Alternatively, peptideassembly can be detected by an increase in donor-acceptor energytransfer between a peptide fragment bearing a cyanine dye of theinvention and a peptide fragment bearing a donor moiety.

When the cleavage agent of interest is a protease, the linker generallyincludes a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. Many protease cleavage sites are known in the art, and theseand other cleavage sites can be included in the linker moiety. See,e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINPRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER′S DISEASE, ed. Masters etal. pp. 190-198 (1994).

Solid Support Immobilized Cyanine Dye Analogues

The cyanine dyes of the invention can be immobilized on substantiallyany polymer, biomolecule, or solid or semi-solid material having anyuseful configuration. Moreover, any conjugate comprising one or morecyanine dye of the invention can be similarly immobilized. When thesupport is a solid or semi-solid, examples of preferred types ofsupports for immobilization of the nucleic acid probe include, but arenot limited to, controlled pore glass, glass plates, polystyrene, avidincoated polystyrene beads, cellulose, nylon, acrylamide gel and activateddextran. These solid supports are preferred because of their chemicalstability, ease of functionalization and well-defined surface area.Solid supports such as, controlled pore glass (CPG, 500 Å, 1000 Å) andnon-swelling high cross-linked polystyrene (1000 Å) are particularlypreferred.

According to the present invention, the surface of a solid support isfunctionalized with a cyanine dye of the invention or a species to whicha cyanine dye of the invention is conjugated. For clarity ofillustration, the following discussion focuses on attaching a reactivecyanine dye of the invention to a solid support. The followingdiscussion is also broadly relevant to attaching to a solid support aspecies that includes within its structure a cyanine dye of theinvention.

The cyanine dyes of the invention are preferably attached to a solidsupport by forming a bond between a reactive group on the cyanine dye ofthe invention and a reactive group on the surface of the solid support,thereby derivatizing the solid support with one or more cyanine dye ofthe invention. Alternatively, the reactive group on the cyanine dye ofthe invention is coupled with a reactive group on a linker arm attachedto the solid support. The bond between the solid support and the cyaninedye of the invention is preferably a covalent bond, although ionic,dative and other such bonds are useful as well. Reactive groups whichcan be used in practicing the present invention are discussed in detailabove and include, for example, amines, hydroxyl groups, carboxylicacids, carboxylic acid derivatives, alkenes, sulfhydryls, siloxanes,etc.

A large number of solid supports appropriate for practicing the presentinvention are available commercially and include, for example, peptidesynthesis resins, both with and without attached amino acids and/orpeptides (e.g., alkoxybenzyl alcohol resin, aminomethyl resin,aminopolystyrene resin, benzhydrylamine resin, etc. (Bachem)),functionalized controlled pore glass (BioSearch Technologies, Inc.), ionexchange media (Aldrich), functionalized membranes (e.g., —COOHmembranes; Asahi Chemical Co., Asahi Glass Co., and Tokuyama Soda Co.),and the like.

Moreover, for applications in which an appropriate solid support is notcommercially available, a wide variety of reaction types are availablefor the functionalization of a solid support surface. For example,supports constructed of a plastic such as polypropylene, can be surfacederivatized by chromic acid oxidation, and subsequently converted tohydroxylated or aminomethylated surfaces. The functionalized support isthen reacted with a cyanine dye of the invention of complementaryreactivity, such as a cyanine dye of the invention active ester, acidchloride or sulfonate ester, for example. Supports made from highlycrosslinked divinylbenzene can be surface derivatized bychloromethylation and subsequent functional group manipulation.Additionally, functionalized substrates can be made from etched, reducedpolytetrafluoroethylene.

When the support is constructed of a siliceous material such as glass,the surface can be derivatized by reacting the surface Si—OH, SiO—H,and/or Si—Si groups with a functionalizing reagent.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by asilicon-modifying reagent such as:(R^(a)O)₃—Si—R^(b)—X^(a)where R^(a) is an alkyl group, such as methyl or ethyl, R^(b) is alinking group between silicon and X^(a), and X^(a) is a reactive groupor a protected reactive group. Silane derivatives having halogens orother leaving groups beside the displayed alkoxy groups are also usefulin the present invention. Exemplary linking groups include those thatinclude substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl groups.

In another preferred embodiment, the reagent used to functionalize thesolid support provides for more than one reactive group per each reagentmolecule. Using reagents, such as the compound below, each reactive siteon the substrate surface is, in essence, “amplified” to two or morefunctional groups:(R^(a)O)₃—Si—R^(b)—(X^(a))_(n)where R^(a) is an alkyl group (e.g., methyl, ethyl), R^(b) is a linkinggroup between silicon and X^(a), X^(a) is a reactive group or aprotected reactive group and n is an integer between 2 and 50, and morepreferably between 2 and 20. The amplification of a cyanine dye of theinvention by its attachment to a silicon-containing substrate isintended to be exemplary of the general concept of amplification. Thisamplification strategy is equally applicable to other aspects of theinvention in which a cyanine dye of the invention is attached to anothermolecule or solid support.

A number of siloxane functionalizing reagents can be used, for example:

-   -   1. Hydroxyalkyl siloxanes (Silylate surface, functionalize with        diborane, and H₂O₂ to oxidize to the alcohol)        -   a. allyl trichlorosilane→→3-hydroxypropyl        -   b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl;    -   2. Diol (dihydroxyalkyl)siloxanes (silylate surface and        hydrolyze to diol)        -   a. (glycidyl            trimethoxysilane→(2,3-dihydroxypropyloxy)propyl;    -   3. Aminoalkyl siloxanes (amines requiring no intermediate        functionalizing step);        -   a. 3-aminopropyl trimethoxysilane→aminopropyl    -   4. Dimeric secondary aminoalkyl siloxanes        -   a.            bis(3-trimethoxysilylpropyl)amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries is available when supportcomponents other than siloxanes are used. Thus, for example alkyl thiols(e.g., self-assembled monolayers), functionalized as discussed above inthe context of siloxane-modifying reagents, can be attached to metalfilms and subsequently reacted with a cyanine dye of the invention toproduce the immobilized compound of the invention.

Exemplary groups of use for R^(b) in the above described embodiments ofthe present invention include, but are not limited to, substituted orunsubstituted alkyl (e.g., substituted or unsubstituted arylalkyl,alkylamino, alkoxy), substituted or unsubstituted aryl (e.g.,substituted or unsubstituted arylalkyl, aryloxy and aryloxyalkyl), acyl(e.g., acylamino, acyloxy), mercapto, saturated or unsaturated cyclichydrocarbyl, substituted or unsubstituted heteroaryl (e.g., substitutedor unsubstituted heteroarylalkyl), substituted or unsubstitutedheterocycloalkyl, and combinations thereof.

Acrylamide-Immobilized Probes

In another exemplary embodiment, a species conjugated to a cyanine dyeof the invention is immobilized within a matrix, such as an acrylamidematrix. In a preferred embodiment, the immobilization is accomplished inconjunction with the “acrydite” process (see, Rehman et al., NucleicAcids Research, 27: 649-655 (1999)). The acrydite method allowsimmobilization of alkene labeled probes within a polymerizedpolyacrylamide network. When target mixes are run past the immobilizedprobe band under electrophoresis conditions, the target nucleic acid iscaptured substantially quantitatively. However, detection of this eventcurrently requires a second probe. In one embodiment, probes bearing acyanine dye of the invention, and/or a fluorophore, are immobilized inan acrylamide matrix and subsequently contacted with the target mix. Byusing fluorescent probes as capture probes, signals from target mixescan be directly detected in real time.

Microarrays

The present invention also provides microarrays including immobilizedcyanine dye of the invention and compounds (e.g., peptides, nucleicacids, bioactive agents, etc.) functionalized with cyanine dye of theinvention. Moreover, the invention provides methods of interrogatingmicroarrays using probes that are functionalized with cyanine dye of theinvention. The immobilized species and the probes are selected fromsubstantially any type of molecule, including, but not limited to, smallmolecules, peptides, enzymes nucleic acids and the like.

Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use of acyanine dye of the invention in conjunction with nucleic acidmicroarrays. This focus is intended to be illustrative and does notlimit the scope of materials with which this aspect of the presentinvention can be practiced.

In another preferred embodiment, the compounds of the present inventionare utilized in a microarray format. The cyanine dye of the invention,or species bearing cyanine dye of the invention can themselves becomponents of a microarray or, alternatively they can be utilized as atool to screen components of a microarray.

Thus, in a preferred embodiment, the present invention provides a methodof screening a microarray. The method includes contacting the members ofthe microarray with, for example, a cyanine dye of the invention-bearingprobe and interrogating the microarray for regions of fluorescence. Inan exemplary embodiment, fluorescent regions are indicative of thepresence of an interaction between the cyanine dye of theinvention-bearing probe and a microarray component.

In another exemplary embodiment, the array comprises an immobilizedcyanine-bearing donor-acceptor energy transfer probe. In thisembodiment, when the probe interacts (e.g., hybridizes) with its target,energy transfer between the cyanine and a quencher moiety is disruptedand the cyanine dye fluoresces. Such arrays are easily prepared andread, and can be designed to give quantitative data. Arrays comprising acyanine-bearing probe are valuable tools for expression analysis andclinical genomic screening.

In another embodiment, the immobilized cyanine-bearing probe is not adonor-acceptor energy transfer probe. A microarray based on such asformat can be used to probe for the presence of interactions between ananalyte and the immobilized probe by, for example, observing thealteration of analyte fluorescence upon interaction between the probeand analyte.

Exemplary microarrays comprise n regions of identical or differentspecies (e.g., nucleic acid sequences, bioactive agents). In a preferredembodiment, n is a number from 2 to 100, more preferably, from 10 to1,000, and more preferably from 100 to 10,000. In a still furtherpreferred embodiment, the n regions are patterned on a substrate as ndistinct locations in a manner that allows the identity of each of the nlocations to be ascertained.

In yet another preferred embodiment, the invention also provides amethod for preparing a microarray of n cyanine-bearing probes. Themethod includes attaching cyanine dye-bearing probes to selected regionsof a substrate. A variety of methods are currently available for makingarrays of biological macromolecules, such as arrays nucleic acidmolecules. The following discussion focuses on the assembly of amicroarray of cyanine-bearing probes, this focus is for reasons ofbrevity and is intended to be illustrative and not limiting.

One method for making ordered arrays of a cyanine-bearing probe on asubstrate is a “dot blot” approach. In this method, a vacuum manifoldtransfers a plurality, e.g., 96, aqueous samples of probes from 3millimeter diameter wells to a substrate. The probe is immobilized onthe porous membrane by baking the membrane or exposing it to UVradiation. A common variant of this procedure is a “slot-blot” method inwhich the wells have highly-elongated oval shapes.

Another technique employed for making ordered arrays of probes uses anarray of pins dipped into the wells, e.g., the 96 wells of a microtiterplate, for transferring an array of samples to a substrate, such as aporous membrane. One array includes pins that are designed to spot amembrane in a staggered fashion, for creating an array of 9216 spots ina 22×22 cm area. See, Lehrach, et al., HYBRIDIZATION FINGERPRINTING INGENOME MAPPING AND SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al,Eds., Cold Springs Harbor Press, pp. 39-81 (1990).

An alternate method of creating ordered arrays of probes is analogous tothat described by Pirrung et al. (U.S. Pat. No. 5,143,854, issued 1992),and also by Fodor et al., (Science, 251: 767-773 (1991)). This methodinvolves synthesizing different probes at different discrete regions ofa particle or other substrate. This method is preferably used withrelatively short probe molecules, e.g., less than 20 bases. A relatedmethod has been described by Southern et al. (Genomics, 13: 1008-1017(1992)).

Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes a method ofmaking an nucleic acid matrix by spotting DNA onto a thin layer ofpolyacrylamide. The spotting is done manually with a micropipette.

The substrate can also be patterned using techniques such asphotolithography (Kleinfield et al., J. Neurosci. 8:4098-120 (1998)),photoetching, chemical etching and microcontact printing (Kumar et al.,Langmuir 10:1498-511 (1994)). Other techniques for forming patterns on asubstrate will be readily apparent to those of skill in the art.

The size and complexity of the pattern on the substrate is limited onlyby the resolution of the technique utilized and the purpose for whichthe pattern is intended. For example, using microcontact printing,features as small as 200 nm are layered onto a substrate. See, Xia, Y.,J. Am. Chem. Soc. 117:3274-75 (1995). Similarly, using photolithography,patterns with features as small as 1 μm are produced. See, Hickman etal., J. Vac. Sci. Technol. 12:607-16 (1994). Patterns which are usefulin the present invention include those which include features such aswells, enclosures, partitions, recesses, inlets, outlets, channels,troughs, diffraction gratings and the like.

In a presently preferred embodiment, the patterning is used to produce asubstrate having a plurality of adjacent wells, indentations or holes tocontain the probes. In general, each of these substrate features isisolated from the other wells by a raised wall or partition and thewells do not readily fluidically communicate. Thus, a particle, reagentor other substance, placed in a particular well remains substantiallyconfined to that well. In another preferred embodiment, the patterningallows the creation of channels through the device whereby an analyte orother substance can enter and/or exit the device.

In another embodiment, the probes are immobilized by “printing” themdirectly onto a substrate or, alternatively, a “lift off” technique canbe utilized. In the lift off technique, a patterned resist is laid ontothe substrate, and a probe is laid down in those areas not covered bythe resist and the resist is subsequently removed. Resists appropriatefor use with the substrates of the present invention are known to thoseof skill in the art. See, for example, Kleinfield et al., J. Neurosci.8:4098-120 (1998). Following removal of the photoresist, a second probe,having a structure different from the first probe can be bonded to thesubstrate on those areas initially covered by the resist. Using thistechnique, substrates with patterns of probes having differentcharacteristics can be produced. Similar substrate configurations areaccessible through microprinting a layer with the desiredcharacteristics directly onto the substrate. See, Mrkish et al. Ann.Rev. Biophys. Biomol. Struct. 25:55-78 (1996).

Linkers

As used herein, the term “linker,” refers to a constituent of aconjugate between a cyanine dye and a carrier molecule. The linker is acomponent of the cyanine dye, the carrier molecule or it is a reactivecross-linking species that reacts with both the carrier molecule and thecyanine dye. The linker groups can be hydrophilic (e.g., tetraethyleneglycol, hexaethylene glycol, polyethylene glycol) or they can behydrophobic (e.g., hexane, decane, etc.). Exemplary linkers includesubstituted or unsubstituted C₆-C₃₀ alkyl groups, polyols (e.g.,glycerol), polyethers (e.g., poly(ethyleneglycol)), polyamines, aminoacids (e.g., polyaminoacids), saccharides (e.g., polysaccharides) andcombinations thereof.

In an exemplary embodiment, the linker joins donor and/or acceptormoieties and other groups to a nucleic acid, peptide or other componentof a probe. In a further exemplary embodiment, using a solid support,the immobilized construct includes a linker attached through the solidsupport and also to the cyanine dye.

In certain embodiments, it is advantageous to have the donor and/oracceptor moieties of the probe attached to a carrier molecule by a groupthat provides flexibility and distances the linked species from thecarrier molecule. Using linker groups, the properties of the donorand/or acceptor moiety is modulated. Properties that are usefullycontrolled include, for example, hydrophobicity, hydrophilicity,surface-activity, the distance of the quencher and/or cyanine dye of theinvention moiety from the other probe components (e.g., carriermolecule) and the distance of the quencher from the cyanine dye of theinvention.

In an exemplary embodiment, the linker serves to distance the cyaninedye of the invention from a nucleic acid to which it is attached.Linkers with this characteristic have several uses. For example, acyanine dye of the invention held too closely to the nucleic acid maynot interact with the quencher group, or it may interact with too low ofan affinity. When a cyanine dye of the invention is itself stericallydemanding, the interaction leading to quenching can be undesirablyweakened, or it may not occur at all, due to a sterically inducedhindering of the approach of the two components.

When the construct comprising the cyanine dye is immobilized byattachment to, for example, a solid support, the construct can alsoinclude a linker moiety placed between the reactive group of the solidsupport and the cyanine analogue, or other probe component bound to thesolid support.

In yet a further embodiment, a linker group used in the probes of theinvention is provided with a group that can be cleaved to release abound moiety, e.g., a cyanine dye of the invention, quencher, minorgroove binder, intercalating moiety, and the like from the polymericcomponent. Many cleaveable groups are known in the art. See, forexample, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshiet al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J.Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986);Browning et al., J. Immunol., 143: 1859-1867 (1989). Moreover a broadrange of cleavable, bifunctional (both homo- and hetero-bifunctional)linker arms is commercially available from suppliers such as Pierce.Exemplary cleaveable groups are those cleaved by light, e.g.,nitrobenzyl derivatives, phenacyl groups, benzoin esters; hydrolysis,e.g., esters, carbonates; changes in pH, etc.

The Methods

In another aspect embodiment, the present invention provides a methodfor detecting a target species in an assay mixture or other sample. Thefollowing discussion is generally relevant to the assays describedherein. This discussion is intended to illustrate the invention byreference to certain preferred embodiments and should not be interpretedas limiting the scope of probes and assay types in which the compoundsof the invention find use. Other assay formats utilizing the compoundsof the invention will be apparent to those of skill in the art.

An exemplary method uses a cyanine dye of the invention or a conjugatethereof to detect a nucleic acid target sequence. The method includes:(a) contacting the target sequence with a detector nucleic acid thatincludes a cyanine dye of the invention and a quencher; (b) hybridizingthe detector nucleic acid to the target sequence, thereby altering theconformation of the detector nucleic acid, causing a change in afluorescence parameter; and (c) detecting the change in the fluorescenceparameter, thereby detecting the nucleic acid target sequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a quencher; andii) a cyanine dye of the invention. Moreover, prior to its hybridizationto a complementary sequence, the detector nucleic acid is preferably ina conformation that allows donor-acceptor energy transfer between thequencher and the cyanine dye of the invention when the fluorophore isexcited. Furthermore, in the methods described in this section, a changein fluorescence is detected as an indication of the presence of thetarget sequence. The change in fluorescence is preferably detected inreal time.

Presently preferred nucleic acid probes do not require the carriermolecule to adopt a secondary structure for the probe to function.Exemplary probes according to this motif include a quencher moiety thatincludes the diazo-linked quenchers described in co-pending, commonlyassigned U.S. patent application Ser. No. 09/567,863 (WO 01/86001) orthe conformationally assisted probes disclosed in U.S. patentapplication Ser. No. 09/591,185.

In other methods described in this section, the detector nucleic acidcan assume substantially any intramolecularly associated secondarystructure, e.g., hairpins, stem-loop structures, pseudoknots, triplehelices and conformationally assisted structures. Moreover, theintramolecularly base-paired secondary structure preferably comprises aportion of the target binding sequence.

In another aspect, the invention provides a method for detectingamplification of a target sequence. The method includes the use of anamplification reaction including the following steps: (a) hybridizingthe target sequence and a detector nucleic acid that includes a cyaninedye of the invention. The detector nucleic acid preferably includes asingle-stranded target binding sequence and an intramolecularlyassociated secondary structure 5′ to the target binding sequence. Atleast a portion of the detector sequence forms a single stranded tailwhich is available for hybridization to the target sequence; (b)extending the hybridized detector nucleic acid on the target sequencewith a polymerase to produce a detector nucleic acid extension productand separating the detector nucleic acid extension product from thetarget sequence; (c) hybridizing a primer to the detector nucleic acidextension product and extending the primer with the polymerase, therebylinearizing the intramolecularly associated secondary structure andproducing a change in a fluorescence parameter; and (d) detecting thechange in the fluorescence parameter, thereby detecting the targetsequence.

In yet a further aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid includes a cyanine dye of theinvention. The method includes: (a) contacting the first nucleic acidwith the second nucleic acid; (b) detecting an alteration in afluorescent property of a member selected from the first nucleic acid,the second nucleic acid and a combination thereof, thereby ascertainingwhether the hybridization occurs.

In general, to determine the concentration of a target molecule, e.g., anucleic acid, it is preferable to first obtain reference data in whichconstant amounts of probe are contacted with varying amounts of target.The fluorescence emission of each of the reference mixtures is used toderive a graph or table in which target concentration is compared tofluorescence emission. For example, a probe that hybridizes to a nucleicacid ligand and has a stem-loop architecture with the 5′ and 3′ terminibeing the sites of quencher and cyanine labeling, can be used to obtainsuch reference data. The value of the fluorescence emission is thencompared with the reference data to obtain the concentration of thetarget in the test mixture.

The cyanine dyes and their conjugates described herein can be used insubstantially any nucleic acid probe format now known or laterdiscovered. For example, the cyanine dyes of the invention can beincorporated into probe motifs, such as Taqman™ probes (Held et al.,Genome Res. 6: 986-994 (1996), Holland et al., Proc. Nat. Acad. Sci. USA88: 7276-7280 (1991), Lee et al., Nucleic Acids Res. 21: 3761-3766(1993)), molecular beacons (Tyagi et al., Nature Biotechnology14:303-308 (1996), Jayasena et al., U.S. Pat. No. 5,989,823, issued Nov.23, 1999)) scorpion probes (Whitcomb et al., Nature Biotechnology 17:804-807 (1999)), sunrise probes (Nazarenko et al., Nucleic Acids Res.25: 2516-2521 (1997)), conformationally assisted probes (Cook, R.,copending and commonly assigned U.S. patent application Ser. No.09/591,185), peptide nucleic acid (PNA)-based light up probes (Kubistaet al., WO 97/45539, December 1997), double-strand specific DNA dyes(Higuchi et al, Bio/Technology 10: 413-417 (1992), Wittwer et al,BioTechniques 22: 130-138 (1997)) and the like. These and other probemotifs with which the present cyanine dyes can be used are reviewed inNONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a cyanine dye of the invention can be used in both in vivoand in vitro enzymatic assays.

Thus, in another aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct that includes acyanine dye of the invention; (b) exciting the fluorophore; and (c)determining a fluorescence property of the sample, wherein the presenceof the enzyme in the sample results in a change in the fluorescenceproperty.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a cyanine dye of theinvention; and iii) a cleavage or assembly recognition site for theenzyme. Moreover, the peptide construct preferably exists in at leastone conformation that allows donor-acceptor energy transfer between thecyanine dye of the invention and the quencher when the fluorophore isexcited.

When the probe is used to detect an enzyme, such as a degradative enzyme(e.g., protease), and a degree of donor-acceptor energy transfer that islower than an expected amount is observed, this is generally indicativeof the presence of an enzyme. The degree of donor-acceptor energytransfer in the sample can be determined, for example, as a function ofthe amount of fluorescence from the donor moiety, the amount offluorescence from the acceptor moiety, the ratio of the amount offluorescence from the donor moiety to the amount of fluorescence fromthe acceptor moiety or the excitation state lifetime of the donormoiety.

The assay also is useful for determining the amount of enzyme in asample. For example, by determining the degree of donor-acceptor energytransfer at a first and second time after contact between the enzyme andthe tandem construct, and determining the difference in the degree ofdonor-acceptor energy transfer. The difference in the degree ofdonor-acceptor energy transfer reflects the amount of enzyme in thesample.

The assay methods also can also be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. Thus, in afurther aspect, the invention provides methods of determining the amountof activity of an enzyme in a sample from an organism. The methodincludes: (a) contacting a sample comprising the enzyme and the compoundwith a peptide construct that includes a cyanine dye of the invention;(b) exciting the fluorophore; and (c) determining a fluorescenceproperty of the sample, wherein the activity of the enzyme in the sampleresults in a change in the fluorescence property. Peptide constructsuseful in this aspect of the invention are substantially similar tothose described immediately above.

In a preferred embodiment, the amount of enzyme activity in the sampleis determined as a function of the degree of donor-acceptor energytransfer in the sample and the amount of activity in the sample iscompared with a standard activity for the same amount of the enzyme. Adifference between the amount of enzyme activity in the sample and thestandard activity indicates that the compound alters the activity of theenzyme.

Representative enzymes with which the present invention can be practicedinclude, for example, trypsin, enterokinase, HIV-1 protease, prohormoneconvertase, interleukin-1b-converting enzyme, adenovirus endopeptidase,cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloidprecursor protein, thrombin, renin, angiotensin-converting enzyme,cathepsin-D and a kininogenase, and proteases in general.

Proteases play essential roles in many disease processes such asAlzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compoundsthat block or enhance their activity have potential as therapeuticagents. Because the normal substrates of peptidases are linear peptidesand because established procedures exist for making non-peptidicanalogs, compounds that affect the activity of proteases are naturalsubjects of combinatorial chemistry. Screening compounds produced bycombinatorial chemistry requires convenient enzymatic assays.

The most convenient assays for proteases are based on donor-acceptorenergy transfer from a donor fluorophore to a quencher placed atopposite ends of a short peptide chain containing the potential cleavagesite (see, Knight C. G., Methods in Enzymol. 248:18-34 (1995)).Proteolysis separates the fluorophore and quencher, resulting inincreased intensity in the emission of the donor fluorophore. Existingprotease assays use short peptide substrates incorporating unnaturalchromophoric amino acids, assembled by solid phase peptide synthesis.

Assays of the invention are also useful for determining andcharacterizing substrate cleavage sequences of proteases or foridentifying proteases, such as orphan proteases. In one embodiment themethod involves the replacement of a defined linker moiety amino acidsequence with one that contains a randomized selection of amino acids. Alibrary of fluorescent cyanine dye probes, wherein the cyanine dyes ofthe invention are linked by a randomized peptide linker moiety, whichcan be generated using recombinant engineering techniques or syntheticchemistry techniques. Screening the members of the library can beaccomplished by measuring a signal related to cleavage, such asdonor-acceptor energy transfer, after contacting the cleavage enzymewith each of the library members of the tandem fluorescent peptideconstruct. A degree of donor-acceptor energy transfer that is lower thanan expected amount indicates the presence of a linker sequence that iscleaved by the enzyme. The degree of donor-acceptor energy transfer inthe sample can be determined, for example, as a function of the amountof fluorescence from the donor moiety, the amount of fluorescence fromthe acceptor donor moiety, or the ratio of the amount of fluorescencefrom the donor moiety to the amount of fluorescence from the acceptormoiety or the excitation state lifetime of the donor moiety.

Multiplex Analyses

In another exemplary embodiment, the cyanine dyes of the invention areutilized as a component of one or more probes used in a multiplex assayfor detecting one or more species in a mixture.

Probes that include a cyanine dye are particularly useful in performingmultiplex-type analyses and assays. In a typical multiplex analysis, twoor more distinct species (or regions of one or more species) aredetected using two or more probes, wherein each of the probes is labeledwith a different fluorophore, quencher or fluorophore/quencher pair.Preferred species used in multiplex analyses relying on donor-acceptorenergy transfer meet at least two criteria: the fluorescent species isbright and spectrally well resolved; and the energy transfer between thefluorescent species and the quencher is efficient.

Thus, in a further embodiment, the invention provides a mixturecomprising at least a first carrier molecule and a second carriermolecule. The first carrier molecule has covalently bound thereto afirst quencher and a first cyanine dye of the invention. An exemplaryquencher has a structure that includes at least three radicals selectedfrom aryl, substituted aryl, heteroaryl, substituted heteroaryl andcombinations thereof. At least two of the radicals are covalently linkedvia an exocyclic diazo bond. The mixture also includes a second carriermolecule. The fluorophore, quencher or both the fluorophore and quencherattached to the second carrier molecule is different than that attachedto the first nucleic acid.

The cyanine dye of the invention allows for the design of multiplexassays in which more than one quencher structure is used in the assay.In one exemplary assay, at least two distinct cyanine dyes of theinvention are used with a common quencher structure. The quencher(s) canbe bound to the same molecule as the cyanine dye of the invention or toa different molecule. Moreover, the carrier molecules of use in aparticular assay system can be the same or different.

In addition those embodiment described above, the present invention alsoprovides a method for detecting or quantifying a particular molecularspecies. The method includes: (a) contacting the species with a mixturesuch as that described above; and (b) detecting a change in afluorescent property of one or more component of the mixture, themolecular species or a combination thereof, thereby detecting orquantifying the molecular species.

Because the present invention provides readily available cyanine dyes,which can be tuned to emit fluorescence of a desired wavelength, thecompounds of the invention are particularly well suited for use inmultiplex applications. Access to cyanine dyes of the invention having arange of emission characteristics allows for the design ofdonor-acceptor energy transfer probes in which the acceptor absorbanceproperties and the emission properties of the cyanine are substantiallymatched, thereby providing a useful level of spectral overlap. Moreover,the cyanine dyes of the invention provide access to probes that emitlight at different wavelengths allows the probes to be spectrallyresolved, which is desirable for multiplex analysis.

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both rhodamine and fluorescein signalis diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:49-53 (1998)) have described the simultaneous use of four differentlylabeled molecular beacons for allele discrimination, and Lee et al.,BioTechniques 27: 342-349 (1999) have described seven color homogenousdetection of six PCR products. The compounds of the invention are of usein such methods.

The quenchers of the present invention can be used in multiplex assaysdesigned to detect and/or quantify substantially any species, including,for example, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

Kits

In another aspect, the present invention provides kits containing one ormore of the cyanine dye of the invention or a conjugate thereof. In oneembodiment, a kit includes a reactive cyanine dye of the invention anddirections for attaching this derivative to another molecule. In anotherembodiment, the kit includes a cyanine-labeled nucleic acid thatoptionally is also labeled with a quencher and directions for using thisnucleic acid in one or more assay format. Other formats for kits will beapparent to those of skill in the art and are within the scope of thepresent invention.

The materials and methods of the present invention are furtherillustrated by the examples that follow. These examples are offered toillustrate, but not to limit the claimed invention.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES Example 1 1.1 Preparation of(1-(ε-carboxypentyl)-1′-(ethyl)-indo)-carbocyanine dye 1

1-(ε-Carboxypentyl)-2,3,3-trimethylindoleninium and1-ethyl-2,3,3-trimethylindoleninium where prepared as described byMujumdar et al., Bioconjugate Chemistry, 4(2):105-111 (1993).

1-(ε-Carboxypentyl)-2,3,3-trimethylindoleninium (2 g, 5.65 mmol) andN,N′-diphenylformamidine (1.5 g, 7.64 mmol) were combined with 1:1acetic acid/acetic anhydride (20 mL) under argon in a 50 mL round-bottomflask. The mixture was heated to light reflux (˜120° C.) and maintainedovernight with stirring. The mixture was cooled and the solvents wereremoved by rotary evaporation, which was followed by high vacuum drying.The residue was redissolved in 1:1 acidic acid/pyridine (100 mL) andtransferred to a 250 mL round-bottom flask. The mixture was heated toreflux with stirring under argon. 1-Ethyl-2,3,3-trimethylindoleninium (2g, 6.3 mmol) was slowly added and the resulting mixture was refluxed for2 h, then cooled and the solvents were removed. The residue wasdissolved in dichloromethane (DCM) and the organic layer was washedrepeatedly with water. The organic layer was separated and dried withmagnesium sulfate (MgSO₄). The dried organic layer was filtered, thesolvent was removed and the residue was vacuum dried to yield a dark redsolid. The crude product was submitted to column chromatography onalumina, eluted with an isocratic mixture of 2% methanol (MeOH), 0.5%pyridine and 97.5% dichloromethane. Yield 2.59 g, 76%

1.2 Preparation of N-hydroxysuccinimide ester 2

Compound 1 (1 g, 1.7 mmol) was dissolved in N,N-dimethylformamide (DMF)(10 mL) in a 25 mL round-bottom flask. 1-Hydroxysuccinimide (0.3 g, 2.6mmol) and dicyclohexylcarbodiimide (DCC) (0.55 g, 2.6 mmol) dissolved inDMF (3 mL) were added drop-wise with stirring. The mixture wasmaintained overnight and filtered to remove the urea. The urea waswashed with DMF. The solvents were removed by rotary evaporation. Theresidue was dissolved in dichloromethane and washed with water 3 times.The organic layer was dried over MgSO₄, filtered and the solventremoved. The residue was vacuum dried to a dark red solid. The product,2, was used without further purification. Yield 0.94 g, 98%

1.2 Preparation of 1-O-(4,4′-dimethoxytrityl)-2-amino-propan-3-ol 3

Serinol (11.2 g, 123 mmol) and methyl trifluoroacetate (32 mL, 318 mmol)in methanol (110 mL) were stirred overnight. The solvent was evaporatedand the residue was co-evaporated twice from pyridine. The resultingresidue was again dissolved in pyridine (50 mL). 4,4′-dimethoxytritylchloride (ChemGenes) (21.0 g, 62 mmol) was dissolved in pyridine (100mL). The resulting solution was added to the serinol-pyridine mixturedrop-wise over 1 hour under argon and the mixture was stirred overnight.The solvents were evaporated and the residue was dissolved in ethylacetate (200 mL). The organic layer was washed with saturated aqueousNaHCO₃ (2×200 mL) and saturated aqueous KHPO₄. The organic layer wasseparated, dried over MgSO₄, gravity filtered and evaporated to half itsoriginal volume. The mixture was purified on silica gel (1200 cc),eluted with 25-60% ethyl acetate+1% triethylamine in petroleum ether.Fractions containing pure mono-DMT adduct, determined by TLC in 35%ethyl acetate+1% triethylamine in petroleum ether, were pooled and thesolvents were evaporated. The product was dissolved in THF (250 mL) and1 M aqueous KOH (250 mL) was added. The mixture was stirred overnight.TLC in 50% ethyl acetate: 1% triethylamine: 49% petroleum ether showedthe hydrolysis was complete. Ethyl acetate (100 mL) was added to thereaction mixture to separate the layers. The aqueous layer was extractedwith ethyl acetate (2×100 mL). The combined organic layers wereevaporated and the residue was dissolved in ethyl acetate (250 mL). Theorganic layer was washed with saturated aqueous KHPO₄ (250 mL), driedover MgSO₄, gravity filtered and evaporated to yield 14.7 g (30%) of 3as a white foam.

1.3 Preparation of Serinol Cyanine Dye 4 by Conjugation of 1 with 3

Compound 1 (2.5 g, 4.2 mmol) was combined withbenzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(Bop) (2.8 g, 6.3 mmol) in dichloromethane (75 mL). N-methylmorpholine(NMM) (1.4 mL) was added and the resulting mixture was stirred for 10minutes. Compound 3 (2.3 g, 5.8 mmol) was dissolved in dichloromethane(50 mL) and the mixture was added drop-wise to mixture including 1. Theresulting mixture was stirred for two hours, and was transferred to aseparatory funnel, where it was washed once with 1M citric acid, oncewith water and once with saturated sodium hydrogen carbonate (aq). Theorganic layer was dried over MgSO₄, filtered and the solvent wasremoved. The residue was vacuum dried to yield dark red solid. The crudeproduct was purified by column chromatography on alumina, eluted with agradient that ranged from 1% MeOH: 0.5% pyridine: 98.5% dichloromethaneto 2.5% MeOH: 0.5% pyridine: 97% dichloromethane. Pure product fractionswhere pooled together evaporated to dryness to give a dark red solid.Yield 1.76 g, 43%

1.4 Preparation of Cyanine Dye 5 by Reaction of 4 with DiglycolicAnhydride

Compound 4 (1.7 g, 1.8 mmol) was dissolved in pyridine (50 mL) in a 100mL round-bottom flask and N-methylimidizole (NMI) (0.1 mL) was addedwith stirring. Diglycolic anhydride (0.45 g, 3.9 mmol) was added and themixture was stirred overnight. The solvent was removed by rotaryevaporation and the residue was dissolved in dichloromethane and washedwith saturated K₂HPO₄ (aq) (4×). The organic layer was removed and driedover MgSO₄, filtered and the solvent was removed. The residue was vacuumdried to give 5 as a dark red, crisp foam. Yield 1.6 g, 80%.

1.5 Preparation of Conjugate Between Cyanine Dye Glycolate and 500 ÅControlled Pore Glass 6

Compound 5 (0.1 g, 0.1 mmol), Bop (0.05 g, 0.1 mmol),1-hydroxybenzotriazole (HOBt) (0.02 g, 0.14 mmol) and acetonitrile (2mL) were combined in a 20 mL scintillation vial. NMM (0.01 mL) was addedand the mixture was stirred for 5 minutes. Aminopropylated 500 Å CPG(2.0 g) was added and the suspension was maintained overnight in aconstant temperature water bath set to 30° C. The CPG was washed oncewith tetrahydrofuran (THF) using a sintered glass funnel. A cappingsolution was made by combining 10% acetic anhydride, 10% NMI and 10%pyridine in THF, and the CPG was contacted with this solution for 30minutes. The CPG was washed repeatedly with THF, dichloromethane andfinally with acetonitrile. The CPG ready for use after air dryingovernight.

1.6 Preparation of 7 by conjugation of 1 with 2-(2-aminoethoxy)-ethanol

Compound 1 (2.5 g, 4.2 mmol) was combined with Bop (2.8 g, 6.3 mmol),NMM (1 mL) in dichloromethane (25 mL) and the mixture was stirred for 10min. 2-(2-Aminoethoxy)-ethanol (0.67 g, 6.4 mmol) was added and theresulting mixture was stirred overnight. The organic layer was washedonce with 0.5M hydrochloric acid (HCL), once with water and once withsaturated sodium bicarbonate (aq). The organic layer was separated,dried over MgSO₄, filtered and the solvent was removed. The resultingresidue was dried under high vacuum before purification. The crudeproduct was submitted to column chromatography on a bed of alumina (4×20cm), eluted with a gradient ranging from 0.5% pyridine: 99.5%dichloromethane to 2.5% MeOH: 0.5% pyridine: 97% dichloromethane. Thefractions containing purified 7 were combined, the solvent was removedand the product vacuum dried overnight. Yield 1.9 g, 66%

1.7 Preparation of the Cyanine Dye Phosphoramidite 8 by Coupling 7 withPhosphane

Phosphane (0.82 g, 2.7 mmol) and 1-H-tetrazole (0.051 g, 0.073 mmol)were dissolved in anhydrous MeCN (12 mL) and mixed for 1 min. Compound 7(1.5 g, 2.2 mmol) was added, the components were mixed and allowed tosit for 2 h. The solvent was removed, the product dissolved indichloromethane and washed once with dilute (2%) sodium hydrogencarbonate solution. The organic layer was separated, dried with MgSO₄,evaporated to dryness and vacuum dried to provide 8 as a dark red foam.Yield 1.5 g 79%

1.8 Preparation of an Oligonucleotide 9 Conjugated to Cyanine Dye 1

Compound 8 was dissolved in anhydrous MeCN at a concentration of 100mg/mL and coupled to the 5′ terminus of a mixed base 15-mer(3′-TTC-GAT-AAG-TCT-AGC-5′) using the micro coupling protocol on aBiosearch 8700 Automated DNA Synthesizer.

1.9 Preparation of Phosphoramidite 10 from Serinol Cyanine Dye 4Preparation of Oligonucleotide 11

Serinol cyanine dye 4 (1.9 g, 1.9 mmol) was azeotropically dried byco-evaporation with anhydrous MeCN. The azeotopically dried material wasfurther dried under high vacuum overnight.

Phosphane (0.7 g, 2.3 mmol) was combined with 1-H-tetrazole (0.042 g,0.06 mmol) in anhydrous MeCN (20 mL), stirred and let sit 1 min.Previously dried 4 (1.9 g, 1.9 mmol) was added, the resulting solutionwas mixed and allowed to sit for 2 h.

Serinol cyanine dye 15-mer oligonucleotide, 11, was prepared asdescribed for 9.

1.10 Preparation of Conjugate 12, Formed Between 1 andN-Hydroxypiperidine

Compound 1 (2.5 g, 4.2 mmol), Bop (2.8 g, 6.3 mmol) and NMM (1 mL) werecombined in dichloromethane (25 mL) and mixed for 10 min.4-Hydroxypiperidine (0.64 g (6.3 mmol) was added and the resultingmixture was stirred overnight. The reaction mixture was washed once with0.5M HCl, once with water and once with saturated sodium bicarbonate(aq). The organic layer was separated, dried over MgSO₄, filtered andthe solvents were evaporated. The crude product was dried under highvacuum before purification. The crude product was purified bychromatography on alumina (4×20 cm) bed, eluted with a gradient rangingfrom 0.5% pyridine: 99.5% dichloromethane to 2.5% MeOH: 0.5% pyridine:97% dichloromethane. Fractions containing the pure 12 were combined, thesolvent was removed and the product was vacuum dried overnight.

1.11 Preparation of Phosphoramidite 13 by Coupling Cyanine Dye 12 withPhosphane Preparation of Oligonucleotide 14

Compound 12 (1.0 g, 1.5 mmol) was azeotropically dried by co-evaporationwith anhydrous MeCN followed by maintaining under high vacuum overnight.

Phosphane (0.5 g, 1.7 mmol) and 1-H-tetrazole (0.03 g, 0.04 mmol) weredissolved in anhydrous MeCN (10 mL) and stirred for 10 min. Previouslydried 11 (1.0 g, 1.5 mmol) was added and the mixture was allowed to sitfor 2 h. The solvent was removed, the product dissolved indichloromethan and washed once with dilute (2%) sodium hydrogencarbonate solution. The organic layer was separated and dried withMgSO₄. The solvent was removed and 13 was dried under high vacuum todark red foam.

Cyanine dye 15-mer oligonucleotide, 14, was prepared as described for 9.

1.12 Preparation of Conjugate 15, Formed Between 1 and 6-Amino-1-Hexanol

Compound 1 (2.5 g, 4.2 mmol), Bop (2.8 g, 6.3 mmol) and NMM (1 mL) werecombined with dichloromethane (25 mL) and mixed for 10 min.6-amino-1-hexanol (0.75 g, 6.3 mmol) was added and mixed overnight. Thereaction mixture was washed once with 0.5M HCl, once with water and oncewith saturated sodium bicarbonate (aq). The organic layer was dried overMgSO₄, filtered and the solvents were evaporated. The crude product wasdried under high vacuum before purification. The crude product waschromatographed on alumina (4×20 cm), eluted with a gradient rangingfrom 0.5% pyridine: 99.5% dichloromethane to 2.5% MeOH: 0.5% pyridine:97% dichloromethane. Fractions containing pure 15 were combined andevaporated to dryness and subsequently maintained under high vacuum.

1.13 Preparation of Phosphoramidite 16 by Coupling Cyanine Dye 15 withPhosphane Preparation of oligonucleotide 17

Compound 15 (1.0 g, 1.5 mmol) was azeotropically dried by co-evaporationwith anhydrous MeCN and kept under high vacuum overnight.

Phosphane (0.5 g, 1.7 mmol) and 1-H-tetrazole (0.03 g, 0.04 mmol) weredissolved in anhydrous MeCN (10 mL) and the mixture was stirred for 10minutes. Previously dried 15 was added and the reaction mixture wasallowed to sit for 2 h. The solvent was removed, the product dissolvedin dichloromethane and washed once with dilute (2%) sodium hydrogencarbonate solution. The organic layer was separated, dried with MgSO₄,the solvent was removed and the residue was vacuum dried to dark redfoam.

Cyanine dye 15-mer oligonucleotide, 17, was prepared as described for 9.

Example 2 2.1 Preparation of(1-(ε-carboxypentyl)-1′-(ethyl)-indo)-dicarbocyanine Dye 18

Compound 18 was prepared as described for compound 1, using N-hexanoicacid-2,3,3-trimethylindolinium (20 g, 72.89 mmol), malonaldehydebis(phenylimine) (20 g 89.97 mmol) and N-ethyl-2,3,3-trimethylindolinium(20 g, 106.2 mmol). Yield: 31 g, 69%.

2.2 Preparation of 1-O-(4,4′-dimethoxytrityl)-2-amino-propan-3-olconjugate 19 of 18

Compound 19 was prepared as described for 2, using compound 18 (3.5 g,7.03 mmol), N-hydroxysuccinimide (0.9 g, 7.82 mmol), dichloromethane(100 mL) and dicyclohexylcarbodiimide (1.5 g, 7.27 mmol). Yield: 95%.

2.3 Preparation of Serinol Cyanine Dye 20 by Conjugation of 18 with 3

Compound 20 was prepared as described for 4, using 19 (6 g, 12.06 mmol),3 (4.5 g 11.44 mmol) Bop (5.3 g 11.98 mmol) and 4-methylmorpholine (2 mL18.19 mmol). Yield: 9.0 g, 75%.

2.4 Preparation of 21 by Reaction of 19 with Diglycolic Anhydride

Glycolate 21 was prepared as described for 5, using diglycolic anhydride(1.0 g, 8.62 mmol), 19 (4 g 4.58 mmol), dimethylethylamine (1 mL, 13.67mmol) and dichloromethane (60 mL). Yield: 7.8 g, 79%.

2.5 Preparation of Conjugate Between Cyanine Dye Glycolate and 500 ÅControlled Pore Glass 22

The cyanine derivatized CPG was prepared as described for 6, using 21(2.4 g 2.34 mmol), BOP (2 g, 4.52 mmol), HOBt (0.5 g, 3.7 mmol), NMM (1mL, 9.1 mmol), aminopropyl CPG (50 g) and acetonitrile (80 mL).

2.6 Preparation of 21 by conjugation of 18 with2-(2-aminoethoxy)-ethanol

Compound 21 was prepared as described for 7, using 18 (18 g, 36.17mmol), Bop (20 g, 45.22 mmol), NMM (5 mL, 45.48 mmol), dichloromethane(200 mL) and 2-(2-aminoethoxy)-ethanol (5 g, 47.56 mmol). Yield: 14 g,55%.

2.7 Preparation of the Cyanine Dye Phosphoramidite 22 by Coupling 6 withPhosphane

Compound 22 was prepared as described for 7, using 15 g (21 mmol) ofcyanine, 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite (8 g,26.54 mmol), 1H-tetrazole (0.5 g, 7.14 mmol) and acetonitrile (100 mL).Yield: 15 g, 78%.

2.8 Preparation of the Serinol Cyanine Dye Phosphoramidite 23 from 20

Compound 23 was prepared as described for compound 10. Thecyanine-labeled 15-mer oligonucleotide was prepared as described for 11.

The present invention provides a novel method of deprotecting andisolating oligonucleotides. While specific examples have been provided,the above description is illustrative and not restrictive. Any one ormore of the features of the previously described embodiments can becombined in any manner with one or more features of any otherembodiments in the present invention. Furthermore, many variations ofthe invention will become apparent to those skilled in the art uponreview of the specification. The scope of the invention should,therefore, be determined not with reference to the above description,but instead should be determined with reference to the appended claimsalong with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

What is claimed is:
 1. A method for detecting a nucleic acid targetsequence, said method comprising: (a) contacting said target sequencewith a detector oligonucleotide comprising a target binding sequence,said detector oligonucleotide having linked thereto, i) a cyanine dye,wherein said cyanine dye is covalently attached to said detectoroligonucleotide by reaction of a reactive functional group on a cyaninedye reagent and a complementary group on said detector oligonucleotide,said cyanine dye reagent having the formula:

wherein R¹ is substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl groups and does not include a carboxylic acidester moiety; R² is substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl; R³ is selected from the group consisting ofH, substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl, or R² and R³, together with the nitrogen to which they areattached, are optionally joined to form a substituted 5-7-memberedheterocycloalkyl; wherein one of R² and R³ comprises said reactivefunctional group; each of L is C(R¹⁷) wherein each R¹⁷ is selected fromthe group consisting of H, halide, substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl, and wherein two adjacentR¹⁷ groups may join to form a ring; n is an integer selected from 1, 2,3 and 4; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are independently selectedfrom the group consisting of substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, halogen, H, NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, andC(Z²)R²²; wherein two substituents selected from R⁴, R⁵, R⁶ and R⁷ onadjacent atoms of the 3H-indolium, together with the carbon atoms towhich they are attached, are optionally joined to form an aromatic ring;and two substituents selected from R⁸, R⁹, R¹⁰ and R¹¹ on adjacent atomsof the indoline, together with the carbon atoms to which they areattached, are optionally joined to form an aromatic ring; Z¹ is O or S;Z² is selected from the group consisting of O, S and NH; R¹⁹ and R²⁰ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl; R²¹is selected from the group consisting of H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl and C(Z³)R²²; whereinZ³ is selected from the group consisting of O, S and NH; R²² is selectedfrom the group consisting of substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl and OR²³, and NR²⁴R²⁵, whereinR²³ is selected from the group consisting of H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²⁶ wherein  R²⁶ is substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl; R²⁴ and R²⁵ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl; or R²⁰and R²¹, together with the nitrogen to which they are attached, areselected from the group consisting of —NHNH₂, —N═C═S and —N═C═O; R¹² isselected from the group consisting of substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl; and R¹³, R¹⁴, R¹⁵ and R¹⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl; andii) a quencher, wherein said detector oligonucleotide is in aconformation allowing donor-acceptor energy transfer between saidcyanine dye and said quencher when said cyanine dye is excited; (b)hybridizing said target binding sequence to said target sequence,thereby altering said conformation of said detector oligonucleotide,causing a change in a fluorescence parameter; and (c) detecting saidchange in said fluorescence parameter, thereby detecting said nucleicacid target sequence.
 2. The method according to claim 1, wherein saidfluorescence parameter is detected in real time.
 3. The method accordingto claim 1, wherein R² comprises said reactive functional group, andsaid reactive functional group is an oxygen-containing reactivefunctional group.
 4. The method according to claim 3, wherein saidoxygen-containing reactive functional group is selected from the groupconsisting of hydroxyl and phosphoramidite.
 5. The method according toclaim 1, wherein R¹ is unsubstituted alkyl; R³ is H; R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, and R¹¹ are each H; R¹² is unsubstituted alkyl; R¹³, R¹⁴, R¹⁵and R¹⁶ are each unsubstituted alkyl; and R¹⁷ is H.
 6. The methodaccording to claim 5, wherein n is 1 or
 2. 7. The method according toclaim 6, wherein R¹ is pentylene; R¹² is ethyl; and R¹³, R¹⁴, R¹⁵ andR¹⁶ are each methyl.
 8. The method according to claim 1, wherein the3′-terminal nucleotide of said detector oligonucleotide is blocked orrendered incapable of extension by a nucleic acid polymerase.
 9. Themethod according to claim 1, wherein said detector oligonucleotide isnot required to adopt a secondary structure for said detectoroligonucleotide to function.
 10. The method according to claim 1,wherein two substituents selected from R⁴, R⁵, R⁶ and R⁷ on adjacentatoms of the 3H-indolium, together with the carbon atoms to which theyare attached, are joined to form an aromatic ring; and two substituentsselected from R⁸, R⁹, R¹⁰ and R¹¹ on adjacent atoms of the indoline,together with the carbon atoms to which they are attached, are joined toform an aromatic ring.
 11. The method according to claim 10, whereinsaid aromatic ring is phenyl.
 12. The method according to claim 1,wherein one of R² and R³ is attached to said detector oligonucleotidethrough a phosphodiester moiety.
 13. A method for detectingamplification of a target sequence comprising, in an amplificationreaction: (a) hybridizing to said target sequence a detectoroligonucleotide comprising a single-stranded target binding sequence andan intramolecularly associated secondary structure 5′ to said targetbinding sequence, wherein at least a portion of said detectoroligonucleotide is a single stranded tail which is available forhybridization to said target sequence, said detector oligonucleotidehaving linked thereto, i) a cyanine dye, wherein said cyanine dye iscovalently attached to said detector oligonucleotide by reaction of areactive functional group on a cyanine dye reagent and a complementarygroup on said detector oligonucleotide, said cyanine dye reagent havingthe formula:

wherein R¹ is substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl groups and does not include a carboxylic acidester moiety; R² is substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl; R³ is selected from the group consisting ofH, substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl, or R² or R³, together with the nitrogen to which they areattached, are optionally joined to form a substituted 5-7-memberedheterocycloalkyl; wherein one of R² and R³ comprises said reactivefunctional group; each of L is C(R¹⁷) wherein each R¹⁷ is a memberindependently selected from H, halide, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl, and wherein twoadjacent R¹⁷ groups may join to form a ring; n is an integer selectedfrom 1, 2, 3 and 4; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H,NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, and C(Z²)R²²; wherein two substituents selectedfrom R⁴, R⁵, R⁶ and R⁷ on adjacent atoms of the 3H-indolium, togetherwith the carbon atoms to which they are attached, are optionally joinedto form an aromatic ring; and two substituents selected from R⁸, R⁹, R¹⁰and R¹¹ on adjacent atoms of the indoline, together with the carbonatoms to which they are attached, are optionally joined to form anaromatic ring; Z¹ is a member selected from O and S; Z² is a memberselected from O, S and NH; R¹⁹ and R²⁰ are independently selected fromthe group consisting of H, substituted or unsubstituted alkyl, andsubstituted or unsubstituted heteroalkyl; R²¹ is selected from the groupconsisting of H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl and C(Z³)R²²; wherein Z³ is selected from thegroup consisting of O, S and NH; R²² is selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl and OR²³, and NR²⁴R²⁵, wherein R²³ is selectedfrom the group consisting of H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and C(O)R²⁶ wherein  R²⁶is substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R²⁴ and R²⁵ are independently selected from the groupconsisting of H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; or R²⁰ and R²¹, together with the nitrogen towhich they are attached, are selected from the group consisting of—NHNH₂, —N═C═S and —N═C═O; R¹² is selected from the group consisting ofsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl; andR¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from the groupconsisting of H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; and ii) a quencher, wherein said detectoroligonucleotide is in a conformation allowing donor-acceptor energytransfer between said cyanine dye and said quencher when said cyaninedye is excited; (b) extending said hybridized detector oligonucleotideon said target sequence with a polymerase to produce a detectoroligonucleotide extension product and separating said detectoroligonucleotide extension product from said target sequence; (c)hybridizing a primer to said detector oligonucleotide extension productand extending the primer with said polymerase, thereby linearizing saidintramolecularly associated secondary structure and producing a changein a fluorescence parameter; and (d) detecting said change in saidfluorescence parameter, thereby detecting said target sequence.
 14. Themethod according to claim 13, wherein said target sequence is amplifiedby a method selected from Strand Displacement Amplification, PolymeraseChain reaction, Self Sustained Sequence Replication, TranscriptionMediated Amplification, and Nucleic Acid Sequence Based Amplification.15. The method according to claim 13, wherein said secondary structurefurther comprises a partially or entirely single-stranded restrictionendonuclease site.
 16. The method according to claim 13, wherein achange in fluorescence intensity is detected.
 17. The method accordingto claim 16, wherein said change in fluorescence intensity is detectedin real-time.
 18. The method according to claim 13, wherein saidintramolecularly associated secondary structure comprises a portion ofsaid target binding sequence.
 19. The method according to claim 13,wherein R² comprises said reactive functional group, and said reactivefunctional group is an oxygen-containing reactive functional group. 20.The method according to claim 19, wherein said oxygen-containingreactive functional group is selected from the group consisting ofhydroxyl and phosphoramidite.
 21. The method according to claim 13,wherein R¹ is unsubstituted alkyl; R³ is H; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,and R¹¹ are each H; R¹² is unsubstituted alkyl; R¹³, R¹⁴, R¹⁵ and R¹⁶are each unsubstituted alkyl; and R¹⁷ is H.
 22. The method according toclaim 21, wherein n is 1 or
 2. 23. The method according to claim 22,wherein R¹ is pentylene; R¹² is ethyl; and R¹³, R¹⁴, R¹⁵ and R¹⁶ areeach methyl.
 24. The method according to claim 13, wherein the3′-terminal nucleotide of said detector oligonucleotide is blocked orrendered incapable of extension by a nucleic acid polymerase.
 25. Themethod according to claim 13, wherein said detector oligonucleotide isnot required to adopt a secondary structure for said detectoroligonucleotide to function.
 26. The method according to claim 13,wherein two substituents selected from R⁴, R⁵, R⁶ and R⁷ on adjacentatoms of the 3H-indolium, together with the carbon atoms to which theyare attached, are joined to form an aromatic ring; and two substituentsselected from R⁸, R⁹, R¹⁰ and R¹¹ on adjacent atoms of the indoline,together with the carbon atoms to which they are attached, are joined toform an aromatic ring.
 27. The method according to claim 26, whereinsaid aromatic ring is phenyl.
 28. The method according to claim 13,wherein said intramolecularly associated secondary structure is selectedfrom the group consisting of hairpins, stem-loop structures,pseudoknots, and triple helices.
 29. The method according to claim 13,wherein one of R² and R³ is attached to said detector oligonucleotidethrough a phosphodiester moiety.
 30. A method for detecting a nucleicacid target sequence, said method comprising: (a) contacting said targetsequence with a detector oligonucleotide comprising a target bindingsequence, said detector oligonucleotide having linked thereto, i) acyanine dye, wherein said cyanine dye is covalently attached to saiddetector oligonucleotide by reaction of a reactive functional group on acyanine dye reagent and a complementary group on said detectoroligonucleotide, said cyanine dye reagent having the formula:

wherein R¹ is a member selected from substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups and does not includea carboxylic acid ester moiety; R² is a member selected from substitutedor unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R³is selected from the group consisting of H, substituted or unsubstitutedalkyl, and substituted or unsubstituted heteroalkyl, or R² and R³,together with the nitrogen to which they are attached, are optionallyjoined to form a substituted 5-7-membered heterocycloalkyl; wherein oneof R² and R³ comprises said reactive functional group; -(L=L)_(n)-L= hasa structure which is selected from the group consisting of:

wherein Z⁴ is H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocycloalkyl; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H,NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, and C(Z²)R²²; wherein Z¹ is a member selectedfrom O and S; Z² is selected from the group consisting of O, S and NH;R¹⁹ and R²⁰ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl; R²¹ is selected from the group consisting of H, substitutedor unsubstituted alkyl and substituted or unsubstituted heteroalkyl andC(Z³)R²²; wherein Z³ is selected from the group consisting of O, S andNH; R²² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR²³,and NR²⁴R²⁵, wherein R²³ is selected from the group consisting of H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and C(O)R²⁶ wherein  R²⁶ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl; R²⁴ andR²⁵ are independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; or R²⁰ and R²¹, together with the nitrogen to which theyare attached, are selected from the group consisting of —NHNH₂, —N═C═Sand —N═C═O; R¹² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; and R¹³,R¹⁴, R¹⁵ and R¹⁶ are independently selected from the group consisting ofH, substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; and ii) a quencher, wherein said detector oligonucleotideis in a conformation allowing donor-acceptor energy transfer betweensaid cyanine dye and said quencher when said cyanine dye is excited; (b)hybridizing said target binding sequence to said target sequence,thereby altering said conformation of said detector oligonucleotide,causing a change in a fluorescence parameter; and (c) detecting saidchange in said fluorescence parameter, thereby detecting said nucleicacid target sequence.
 31. The method according to claim 30, wherein R²comprises said reactive functional group, and said reactive functionalgroup is an oxygen-containing reactive functional group.
 32. The methodaccording to claim 31, wherein said oxygen-containing reactivefunctional group is selected from the group consisting of hydroxyl andphosphoramidite.
 33. The method according to claim 30, wherein the3′-terminal nucleotide of said detector oligonucleotide is blocked orrendered incapable of extension by a nucleic acid polymerase.
 34. Themethod according to claim 30, wherein said detector oligonucleotide isnot required to adopt a secondary structure for said detectoroligonucleotide to function.
 35. The method according to claim 30,wherein one of R² and R³ is attached to said detector oligonucleotidethrough a phosphodiester moiety.
 36. A method for detectingamplification of a target sequence comprising, in an amplificationreaction: (a) hybridizing to said target sequence a detectoroligonucleotide comprising a single-stranded target binding sequence andan intramolecularly associated secondary structure 5′ to said targetbinding sequence, wherein at least a portion of said detectoroligonucleotide is a single stranded tail which is available forhybridization to said target sequence, said detector oligonucleotidehaving linked thereto, i) a cyanine dye, wherein said cyanine dye iscovalently attached to said detector oligonucleotide by reaction of areactive functional group on a cyanine dye reagent and a complementarygroup on said detector oligonucleotide, said cyanine dye reagent havingthe formula:

wherein R¹ is a member selected from substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups and does notcomprise a carboxylic acid ester moiety; R² is a member selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; R³ is selected from the group consisting of H, substitutedor unsubstituted alkyl, and substituted or unsubstituted heteroalkyl, orR² and R³, together with the nitrogen to which they are attached, areoptionally joined to form a substituted 5-7-membered heterocycloalkyl;wherein one of R² and R³ comprises said reactive functional group;-(L=L)_(n)-L= has a structure which is a member selected from:

wherein Z⁴ is H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocycloalkyl; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H,NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, and C(Z²)R²²; wherein Z¹ is a member selectedfrom O and S; Z² is selected from the group consisting of O, S and NH;R¹⁹ and R²⁰ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl; R²¹ is a member selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl andC(Z³)R²²; wherein Z³ is selected from the group consisting of O, S andNH; R²² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR²³,and NR²⁴R²⁵, wherein R²³ is selected from the group consisting of H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and C(O)R²⁶ wherein  R²⁶ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl; R²⁴ andR²⁵ are independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; or R²⁰ and R²¹, together with the nitrogen to which theyare attached, are selected from the group consisting of —NHNH₂, —N═C═Sand —N═C═O; R¹² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; and R¹³,R¹⁴, R¹⁵ and R¹⁶ are independently selected from the group consisting ofH, substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; and ii) a quencher, wherein said detector oligonucleotideis in a conformation allowing donor-acceptor energy transfer betweensaid cyanine dye and said quencher when said cyanine dye is excited; (b)extending said hybridized detector oligonucleotide on said targetsequence with a polymerase to produce a detector oligonucleotideextension product and separating said detector oligonucleotide extensionproduct from said target sequence; (c) hybridizing a primer to saiddetector oligonucleotide extension product and extending the primer withsaid polymerase, thereby linearizing said intramolecularly associatedsecondary structure and producing a change in a fluorescence parameter;and (d) detecting said change in said fluorescence parameter, therebydetecting said target sequence.
 37. The method according to claim 36,wherein R² comprises said reactive functional group, and said reactivefunctional group is an oxygen-containing reactive functional group. 38.The method according to claim 37, wherein said oxygen-containingreactive functional group is selected from the group consisting ofhydroxyl and phosphoramidite.
 39. The method according to claim 36,wherein the 3′-terminal nucleotide of said detector oligonucleotide isblocked or rendered incapable of extension by a nucleic acid polymerase.40. The method according to claim 36, wherein said detectoroligonucleotide is not required to adopt a secondary structure for saiddetector oligonucleotide to function.
 41. The method according to claim36, wherein said intramolecularly associated secondary structure isselected from the group consisting of hairpins, stem-loop structures,pseudoknots, and triple helices.
 42. The method according to claim 36,wherein one of R² and R³ is attached to said detector oligonucleotidethrough a phosphodiester moiety.
 43. A method for detecting orquantifying a nucleic acid, said method comprising: (a) contacting thenucleic acid with a mixture comprising at least a first carrier moleculeand a second carrier molecule, wherein said first carrier molecule hascovalently bound thereto a first quencher and a first fluorophore,wherein said first fluorophore is a cyanine dye, wherein said cyaninedye is covalently attached to said first carrier molecule by reaction ofa reactive functional group on a cyanine dye reagent and a complementarygroup on said first carrier molecule, said cyanine dye reagent havingthe formula:

wherein R¹ is a member selected from substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups and does not includea carboxylic acid ester moiety; R² is a member selected from substitutedor unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R³is selected from the group consisting of H, substituted or unsubstitutedalkyl, and substituted or unsubstituted heteroalkyl, or R² and R³,together with the nitrogen to which they are attached, are optionallyjoined to form a substituted 5-7-membered heterocycloalkyl; wherein oneof R² and R³ comprises said reactive functional group; each of L isC(R¹⁷) wherein each R¹⁷ is a member independently selected from H,halide, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl, and wherein two adjacent R¹⁷ groups may jointo form a ring; n is an integer selected from 1, 2, 3 and 4; R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are independently selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl, halogen, H, NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, and C(Z²)R²²;wherein two substituents selected from R⁴, R⁵, R⁶ and R⁷ on adjacentatoms of the 3H-indolium, together with the carbon atoms to which theyare attached, are joined to form an aromatic ring; and two substituentsselected from R⁸, R⁹, R¹⁰ and R¹¹ on adjacent atoms of the indoline,together with the carbon atoms to which they are attached, are joined toform an aromatic ring Z¹ is a member selected from O and S; Z² isselected from the group consisting of O, S and NH; R¹⁹ and R²⁰ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, and substituted or unsubstituted heteroalkyl; R²¹is selected from the group consisting of H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl and C(Z³)R²²; whereinZ³ is selected from the group consisting of O, S and NH; R²² is selectedfrom the group consisting of substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl and OR²³, and NR²⁴R²⁵, whereinR²³ is selected from the group consisting of H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²⁶ wherein  R²⁶ is a member selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; R²⁴ and R²⁵ are independently selected from the groupconsisting of H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; or R²⁰ and R²¹, together with the nitrogen towhich they are attached, are selected from the group consisting of—NHNH₂, —N═C═S and —N═C═O; R¹² is selected from the group consisting ofsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl; andR¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from the groupconsisting of H, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; and said second carrier molecule has attachedthereto a member selected from a second fluorophore, a second quencherand a combination thereof, wherein said second fluorophore, said secondquencher, or said second fluorophore and said second quencher isdifferent from said first fluorophore and said first quencher,respectively; and (b) detecting a change in a fluorescent property ofone or more component of the mixture, the nucleic acid or a combinationthereof, thereby detecting or quantifying the nucleic acid.
 44. Themethod according to claim 43, wherein R² comprises said reactivefunctional group, and said reactive functional group is anoxygen-containing reactive functional group.
 45. The method according toclaim 44, wherein said oxygen-containing reactive functional group isselected from the group consisting of hydroxyl and phosphoramidite. 46.The method according to claim 43, wherein R¹ is unsubstituted alkyl; R³is H; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are each H; R¹² isunsubstituted alkyl; R¹³, R¹⁴, R¹⁵ and R¹⁶ are each unsubstituted alkyl;and R¹⁷ is H.
 47. The method according to claim 46, wherein n is 1 or 2.48. The method according to claim 47, wherein R¹ is pentylene; R¹² isethyl; and R¹³, R¹⁴, R¹⁵ and R¹⁶ are each methyl.
 49. The methodaccording to claim 43, wherein said first carrier molecule is a nucleicacid probe, wherein the 3′-terminal nucleotide of said nucleic acidprobe is blocked or rendered incapable of extension by a nucleic acidpolymerase.
 50. The method according to claim 43, wherein said firstcarrier molecule is a nucleic acid probe, wherein said nucleic acidprobe does not require said first carrier molecule to adopt a secondarystructure for said nucleic acid probe to function.
 51. The methodaccording to claim 43, wherein said aromatic ring is phenyl.
 52. Themethod according to claim 43, wherein one of R² and R³ is attached tosaid first carrier molecule through a phosphodiester moiety.
 53. Amethod for detecting or quantifying a nucleic acid, said methodcomprising: (a) contacting the nucleic acid with a mixture comprising atleast a first carrier molecule and a second carrier molecule, whereinsaid first carrier molecule has covalently bound thereto a firstquencher and a first fluorophore, wherein said first fluorophore is acyanine dye, wherein said cyanine dye is covalently attached to saidfirst carrier molecule by reaction of a reactive functional group on acyanine dye reagent and a complementary group on said first carriermolecule, said cyanine dye reagent having the formula:

wherein R¹ is a member selected from substituted or unsubstituted alkyl,and substituted or unsubstituted heteroalkyl groups and does not includea carboxylic acid ester moiety; R² is a member selected from substitutedor unsubstituted alkyl and substituted or unsubstituted heteroalkyl; R³is selected from the group consisting of H, substituted or unsubstitutedalkyl, and substituted or unsubstituted heteroalkyl, or R² and R³,together with the nitrogen to which they are attached, are optionallyjoined to form a substituted 5-7-membered heterocycloalkyl; wherein oneof R² and R³ comprises said reactive functional group; -(L=L)_(n)-L= hasa structure which is a member selected from:

wherein Z⁴ is H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocycloalkyl; R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, H,NO₂, CN, Z¹R¹⁹, NR²⁰R²¹, and C(Z²)R²²; wherein Z¹ is a member selectedfrom O and S; Z² is selected from the group consisting of O, S and NH;R¹⁹ and R²⁰ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl; R²¹ is a member selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl andC(Z³)R²²; wherein Z³ is elected from the group consisting of O S and NH;R²² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl and OR²³,and NR²⁴R²⁵, wherein R²³ is selected from the group consisting of H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and C(O)R²⁶ wherein R²⁶ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl; R²⁴ andR²⁵ are independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; or R²⁰ and R²¹, together with the nitrogen to which theyare attached, are selected from the group consisting of —NHNH₂, —N═C═Sand —N═C═O; R¹² is selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl; and R¹³,R¹⁴, R¹⁵ and R¹⁶ are independently selected from the group consisting ofH, substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; said second carrier molecule has attached thereto a memberselected from a second fluorophore, a second quencher and a combinationthereof, wherein said second fluorophore, said second quencher, or saidsecond fluorophore and said second quencher is different from said firstfluorophore and said first quencher, respectively; and (b) detecting achange in a fluorescent property of one or more component of themixture, the nucleic acid or a combination thereof, thereby detecting orquantifying the nucleic acid.
 54. The method according to claim 53,wherein R² comprises said reactive functional group, and said reactivefunctional group is an oxygen-containing reactive functional group. 55.The method according to claim 54, wherein said oxygen-containingreactive functional group is selected from the group consisting ofhydroxyl and phosphoramidite.
 56. The method according to claim 53,wherein said first carrier molecule is a nucleic acid probe, wherein the3′-terminal nucleotide of said nucleic acid probe is blocked or renderedincapable of extension by a nucleic acid polymerase.
 57. The methodaccording to claim 53, wherein said first carrier molecule is a nucleicacid probe, wherein said nucleic acid probe does not require said firstcarrier molecule to adopt a secondary structure for said nucleic acidprobe to function.
 58. The method according to claim 53, wherein one ofR² and R³ is attached to said first carrier molecule through aphosphodiester moiety.